Methods of preventing bacterial infections with florfenicol-type antibiotics

ABSTRACT

The present invention relates to novel florfenicol compounds having the chemical structure:  
                 
wherein the compounds are useful for the treatment and/or prevention of bacterial infections in a broad range of patients such as, without limitation, birds, fish, shellfish and mammals.

RELATED APPLICATIONS

This Application is a continuation-in-part of U.S. patent applicationSer. No. 10/094,688, filed Mar. 8, 2002 and entitled “NovelFlorfenicol-type Antibiotics.” The '688 application is incorporated asif fully set forth herein.

FIELD OF THE INVENTION

The present invention relates to the fields of organic chemistry,pharmaceutical chemistry, biochemistry and medicine. In particular, itrelates to novel florfenicol-type antibiotics.

BACKGROUND OF THE INVENTION

Florfenicol is a broad spectrum antibiotic with activity against manygram-negative and gram-positive bacteria. Florfenicol is useful for theprevention and treatment of bacterial infections due to susceptiblepathogens in birds, reptiles, fish, shellfish and mammals. One of itsprimary uses is in the treatment of pneumonia and associated respiratoryinfections in cattle (often referred to generically as BovineRespiratory Disease or BRD) caused by Mannhemia haemolytica, Pasturellamultocida and(or) Haemophilus somnus. It is also indicated in thetreatment of pododermatitis in cattle caused by Fusobacteriumnecrophorum and Bacterioides melaninogenicus, swine respiratory diseasecaused by Pasteurella multocida, Actinobacillus pleuropneumoniae,Streptococcus suis, Salmonella cholerasuis and(or) Mycoplasma spp.,colibacillosis in chickens caused by Escherichia coli, entericsepticemia in catfish caused by Edwardsiella ictaluri, and furunculosisin salmon caused by Aeromonas salmonicida. Other genera of bacteria thathave exhibited susceptibility to florfenicol include. Enterobacter,Klebsiella, Staphylococcus, Enterococcus, Bordetella, Proteus, andShigella. In particular, chloramphenicol resistant strains of organismssuch as K. pneumoniae, E. cloacae, S. typhus and E. coli are susceptibleto florfenicol.

Florfenicol is a structural analog of thiamphenicol, which in turn is aderivative of chloramphenicol in which the aromatic nitro group, whichnitro group has been implicated in

chloramphenicol-induced, non-dose related irreversible aplastic anemiain humans, is replaced with a methylsulfonyl group. Florfenicol has afluorine atom in place of the primary hydroxyl group of chloramphenicoland thiamphenicol. This renders florfenicol less susceptible todeactivation by bacteria containing the plasmid-encoded enzyme,chloramphenicol acetyl transferase (CAT), which acetylates the primaryhydroxyl group of chloramphenicol and thiamphenicol, thereby preventingthem from binding to ribosomal subunits of susceptible bacteria.Ribosomal binding is the primary mechanism of action of thechloramphenicol antibiotics and results in inhibition of peptidyltransferase, which is responsible for the transfer of amino acids togrowing peptide chains and subsequent protein formation in bacteria.Nonetheless, compounds having the primary hydroxyl group do have utilityin the treatment of bacterial infections, as evidenced by the continuinguse of chloramphenicol and thiampheniol throughout the world.

In recent years, a number of bacterial genera and species have begun toexhibit some resistance to florfenicol. For example, resistance has beenobserved in Salmonella species (Bolton, L. F., et al., Clin. Microbiol.1999, 37, 1348), E. coli (Keyes, K., et al., Antimicrob. AgentsChemother., 2000, 44, 421.), Klebsiella pneumoniae (Cloeckaert, A., etal., Antimicrob. Agents Chemother., 2001, 45, 2381), and in theaquacultural pathogen, Photobacterium damselae subsp. piscicida(formerly Pasteurella piscicida) (Kim, E., et al., Microbiol. Immunol.,1996, 40, 665). This resistance has been traced to a highly conservedgene (flo) that produces an antibiotic efflux pump (Flo).

The emergence, and threatened spread, of resistance to florfenicol hasfostered the need for new antibiotics that retain or exceed the activityof florfenicol, maintain its imperviousness to the CAT enzyme and, inaddition, are not substrates for the Flo efflux pump. The compounds ofthe present invention are such antibiotics.

SUMMARY

Thus, an embodiment of this invention is a compound having the chemicalformula:

wherein:

-   R¹ is selected from the group consisting of —OH and —F;-   R² and R³ are independently selected from the group consisting of    hydrogen, (1C-4C)alkyl, halo, —CF₃, —NH₂, —CN and N₃;-   R⁴ is selected from the group consisting of: —C(═R⁵)R⁶,    -   wherein:    -   R⁵ is selected from the group consisting of oxygen, N—C≡N, and        NOR⁷,        -   wherein:        -   R⁷ is selected from the group consisting of hydrogen, alkyl,            aryl, heteroaryl, alicyclic and heteroalicyclic;    -   R⁶ is selected from the group consisting of hydrogen,        (1C-4C)alkyl, (3C-6C)cycloalkyl, (1C-4C)alkoxy, aryl,        heteroaryl, alicyclic and heteroalicyclic;        wherein:-   A¹ is carbon or nitrogen;-   A², A³, A⁴ and A⁵ are independently selected from the group    consisting of carbon, nitrogen, oxygen and sulfur, provided that at    least one of A¹-A⁵ is not carbon, that the total number of nitrogen,    oxygen and sulfur atom in the ring does not exceed 4 and that the    ring is aromatic;-   carbon atoms in the ring are independently substituted with an    entity selected from the group consisting of hydrogen, (1C-4C)alkyl,    (3C-6C)cycloalkyl, (1C-4C)alkylO—, —CF₃, —OH, —CN, halo,    (1C-4C)alkylS(O)—, (1C-4C)alkylS(O)₂—, NH₂SO₂—, (1C-4C)alkylNHSO₂—,    ((1C-4C)alkyl)₂NSO₂—, —NH₂, (1C-4C)alkylNH—, ((1C-4C)alkyl)₂N—,    (1C-4C)alkylSO₂NH—, (1C-4C)alkylC(O)—, (3C-6C)cycloalkylC(O)—,    (1C-4C)alkylOC(O)—, (1C-4C)alkylC(O)NH—, —C(O)NH₂,    (1C-4C)alkylNHC(O)— and ((1C-4C)alkyl)₂NC(O)—, wherein any of the    alkyl groups in any of the substituents may optionally be    substituted with a group selected from halo and —OH;-   if A¹ is carbon and the ring does not contain oxygen or sulfur, one    of the nitrogen atoms may optionally be substituted with an entity    selected from the group consisting of (1C-4C)alkyl,    (1C-4C)alkylS(O)₂— and —NH₂;    wherein:-   A⁶, A⁷, A⁸, A⁹ and A¹⁰ are independently selected from the group    consisting of carbon, nitrogen and    provided that only one of A⁶-A¹⁰ at a time can be    carbon atoms in the ring are independently substituted with an    entity selected from the group consisting of hydrogen, (1C-4C)alkyl,    (3C-6C)cycloalkyl, (1C-4C)alkylO—, —CF₃, —OH, —CN, halo,    (1C-4C)alkylS(O)—, (1C-4C)alkylS(O)2—, NH₂SO₂—, (1C-4C)alkylNHSO₂—,    ((1C-4C)alkyl)₂NSO₂—, —NH₂, (1C-4C)alkylNH—, ((1C-4C)alkyl)₂N—,    (1C-4C)alkylSO₂NH—, (1C-4C)alkylC(O)—, (3C-6C)cycloalkylC(O)—,    (1C-4C)alkylOC(O)—, (1C-4C)alkylC(O)NH—, —C(O)NH₂,    (1C-4C)alkylNHC(O)—, ((1C-4C)alkyl)₂NC(O)— and —OCH₂O—, the oxygen    atoms in the —OCH₂O— substituent being bonded to adjacent ring    carbon atoms, wherein any of the alkyl groups in any of the    substituents may optionally be substituted with a group selected    from halo and —OH;-   R⁸ is hydrogen in all compounds, except when R² and R³ are both F,    in which case R⁸is hydrogen or F; and,-   the compound is either a racemate having the relative    stereochemistry shown or is substantially enantiomerically pure and    has the absolute stereochemistry shown.

In an embodiment of this invention, R¹ is —F.

In an embodiment of this invention, R² and R³ are independently selectedfrom the group consisting of Cl and F.

In an embodiment of this invention, R⁸ is hydrogen.

In an embodiment of this invention, R⁴ is —C(═R⁵)R⁶ wherein R⁵ and R⁶are as defined above.

In an embodiment of this invention, R⁴ is CH₃C(O)—.

In an embodiment of this invention, R⁴ is

wherein:

-   A⁶, A⁷, A⁸, A⁹ and A¹⁰ are as defined above, and,-   any of A⁶-A¹⁰ that is carbon is substituted with an entity selected    from the group consisting of hydrogen, —NH₂, halo-, —CN,    (1C-4C)alkyl-, (1C-4C)alkylC(O)—, (1C-4C)alkylS(O)—,    (1C-4C)alkylS(O)₂—, NH₂SO₂—, (1C-4C)alkylSO₂NH—, (1C-4C)alkylNHSO₂—,    ((1C-4C)alkyl)₂NSO₂—, wherein any of the alkyl groups in any of the    substituents may optionally be substituted with halo or —OH.

In an embodiment of this invention, R⁴ is

and one, two or three of A⁶-A¹⁰ is/are nitrogen; and,

-   one or two of the remaining carbon atoms in the ring is/are    optionally substituted with —NH₂, all other carbon atoms in the ring    being unsubstituted.

In a presently preferred embodiment of this invention, R⁴ is selectedfrom the group consisting of:

In an embodiment of this invention, R⁴ is

as defined above.

In an embodiment of this invention, R⁴ is

and all carbon atoms and nitrogen atoms are unsubstituted.

In an embodiment of this invention, R⁴ is

and one of A²-A⁵ that is carbon is substituted with an —NH₂ group, allother carbon and, if applicable, nitrogen atoms in the ring beingunsubstituted.

In a presently preferred embodiment of this invention, R⁴ is selectedfrom the group consisting of:

A presently preferred embodiment of this invention is a compoundselected from the group consisting of:

wherein the compound is either a racemate having the relativestereochemistry shown or is substantially enantiomerically pure and hasthe absolute stereochemistry shown.

A presently particularly preferred embodiment of this invention is acompound selected from the group consisting of:

wherein the compound is either a racemate having the relativestereochemistry shown or is substantially enantiomerically pure and hasthe absolute stereochemistry shown.

In another particularly preferred embodiment of this invention, thecompound herein is substantially enantiomerically pure and has a1-(R)-2-(S) absolute configuration.

An embodiment of this invention is a method of treating or preventing abacterial infection, comprising administering to a patient in needthereof a pharmaceutically effective amount of a compound hereof.

In an embodiment of this invention, the bacterial infection is caused bya bacteria of the genus Pasteurella, Haemophilus, Fusobacterium,Bacterioides, Aeromonas, Enterobacter, Escherichia, Klebsiella,Salmonella, Shigella, Actinobacillus, Streptococcus, Mycoplasma,Edwardsiella, Staphylococcus, Enterococcus, Bordetella, Proteus, orMannheimia.

In an embodiment of this invention the bacterial infection is caused byMannhemia haemolytica, Pasteurella multocida, Haemophilus somnus,Fusobacterium necrophorum, Bacterioides melaninogenicus, Actinobacilluspleuropneumoniae, Streptococcus suis, Salmonella cholerasuis, Mycoplasmabovis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasmagallisepticum, Edwardsiella ictaluri, Escherichia coli, Enterobactercloacae, Staphylococcus aureus, Staphylococcus intermedius, Enterococcusfaecalis, Enterococcus faecium, Klebsiella pneumoniae, Klebsiellaoxytoca, Enterobacter cloacae, Proteus mirabilis, or Aeromonassalmonicida.

DETAILED DESCRIPTION OF THE INVENTION

Brief Description of the Tables

Table 1 shows structures of representative compounds of this invention.The table and the compounds therein are not intended, nor should they beconstrued, to limit this invention in any manner whatsoever.

Table 2 is a list of the microorganisms against which the compounds ofthis invention were tested. The list is not intended, nor should it beconstrued, to limit the scope of this invention in any mannerwhatsoever.

DEFINITIONS

As used herein, “halo” refers to fluorine, chlorine, bromine or iodine.

As used herein, “alkyl” refers to a saturated (containing no multiplebonds) aliphatic (no delocalized π-electron system), hydrocarbon(containing, if unsubstituted, only carbon and hydrogen). Thedesignation (n₁C-n₂C)alkyl, wherein n₁ and n₂ are integers from 1-6,refers to a straight chain or branched chain alkyl comprising from n₁ ton₂ carbon atoms. For example, (1C-4C)alkyl refers to CH₃—, CH₃CH₂—,CH₃CH₂CH₂—, CH₃CH(CH₃)—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— or (CH₃)₃C—. Thealkyl group may be unsubstituted or substituted with one or moremoieties selected from the group consisting of halo, —OH, OCH₃, and—C≡N.

As used herein, “cycloalkyl” refers to an all-carbon cyclic or fusedmulticyclic ring, which, although it may contain one or more doublebonds, maintains an essentially aliphatic character; that is, the doublebonds do not interact to form a delocalized π-electron system around thering. For the purposes of this invention, the ring may contain up to 7carbon atoms. The designation (3C-6C)cycloalkyl refers to 3, 4, 5, and6-member all-carbon-atom rings. As used herein, “fused” means that twocycloalkyl groups share at least one ring atom between them. Thus, suchcompounds as spiro[4.4]nonane are considered “fused” for the purposes ofthis invention. More commonly, fused rings share two adjacent ringcarbon atoms. An example of such a fused system is decalin. A cycloalkylring may be unsubstituted or substituted with a moiety selected from thegroup consisting of —OH, —OCH₃, halo and —C≡N.

As used herein, “aryl” refers to an all-carbon 6-member ring or twofused six-member rings, the ring or fused rings having a delocalizedπ-electron system. By “fused” is meant that each ring of the systemshares two adjacent ring carbon atoms with at least one other ring. Anaryl ring may be unsubstituted or substituted with one or more moietiesselected from the group consisting of —OH, —OCH₃, halo and —C≡N.

As used herein, “heteroaryl” refers to a five-member or six-member ringor to two rings, i.e., two 5-member, two six-member or a five- and asix-member ring fused together wherein the ring or fused ring has adelocalized π-electron system. If a ring is six-membered, it mustconsist of carbon and nitrogen only and may contain from one to fournitrogen atoms. If a ring is five-membered, it must contain onenitrogen, oxygen or sulfur atom and may contain one, two or threeadditional nitrogen atoms. A five-member ring with a circle in thecenter indicates that the ring is heteroaromatic. The circle is used toemphasize the fact that the location of the double bonds thatparticipate in making the ring heteroaromatic is not static, rather itis dependent on the nature of the atoms forming the ring, i.e., whetherthey are carbon, nitrogen, oxygen or sulfur and what groups, if any, arebonded to them. The actual structure of any five-member heteroaromaticwill be immediately apparent to those skilled in the art once the ringatoms are designated. With regard to heteroaromatic groups, the termfused has the same meaning as in -the case of aryl groups. A heteroarylgroup may be unsubstituted or substituted with any of the moietiesdescribed above with regard to aryl groups.

As used herein, “heteroalicyclic” refers to a cyclic or fused cyclicring system containing atoms selected from the group consisting ofcarbon, nitrogen, oxygen and sulfur but no delocalized π-electronsystem. “Fused” has the same meaning set forth above with regard tocycloalkyl rings. Likewise, a heteroalicyclic ring may be unsubstitutedor substituted with the same moieties described above for cycloalkylrings.

Whenever a ring carbon atom is stated to be “unsubstituted,” it isunderstood that any unfilled valences are in fact occupied by hydrogenatoms. Likewise, if a ring nitrogen atom is capable of being furthersubstituted and it is stated to be unsubstituted, it means that thenitrogen is bonded to a hydrogen atom.

As used herein, “relative stereochemistry” refers to the positioning inspace of substituents relative to one another.

As used herein, “absolute stereochemistry” refers to the exactpositioning of substitutents in three-dimensional space as determined bythe Cahn-Ingold-Prelog rules, the application of which are well-known tothose skilled in the art.

As used herein, an “enantiomer” refers to one of the two absolutestereochemical configurations of a molecule that rotates plane polarizedlight in one direction or the other (i.e.; counterclockwise from itsoriginal axis, conventionally called “left,” or clockwise,conventionally referred to as “right”). By “substantiallyenantiomerically pure” is meant that the compound consists of greaterthan 90% of the one enantiomer, preferably greater than 95%, and mostpreferably greater than 99%.

As used herein, a “racemate” refers to a 1:1 mixture of the twoenantiomers of a compound. Racemic mixtures are designated by a (±)indicator. Substantially enantiomerically pure compounds are shownwithout the indicator.

As used herein, “patient” refers to birds, reptiles, fish, shellfish,and mammals. In particular it refers to birds such as, withoutlimitation, chickens and turkeys, fish such as, without limitation,salmon, trout, catfish and yellowtail, mammals such as, withoutlimitation, cats, dogs, rabbits, sheep, cattle, pigs, horses and goatsand to human beings.

Discussion

The compounds of this invention are expected to be useful for thetreatment of bacterial infections in patients.

Compounds

The compounds of the present invention are set forth generally in theSummary, above. Exemplary compounds of this invention are shown inTable 1. Neither the table nor the compounds shown therein are intended,or are to be construed, as limiting the scope of this invention in anymanner whatsoever. TABLE 1 Compound # Structure 28

29

41

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

71

72

73

74

75

76

77

78

79

80

81

82

84

85

86

91

92

93

94

95

97

98

100

101

106

107

109

110

111

112

113

114

115

116

117

118

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

Syntheses

Intermediates were prepared in enantiomerically pure form, starting withcommercially available chloramphenicol base (1), or as racemic mixturesby condensation of ρ-bromobenzaldehyde with glycine under basicconditions. For the enantiomerically pure synthesis of intermediatessuch as 9, chloramphenicol base was converted to compound 3 (Rebstock,M. C., et al., J. Am, Chem. Soc., 1949, 71, 2458; Evans, D. D., et al.,J. Chem. Soc., 1954, 1687; Morris, D. S. and Smith, S. D., J. Chem.Soc., 1954, 1680). Compound 3 was subjected to the Sandmeyer reactionafter which the acetate protecting group was removed under acidicconditions to provide enantiomerically pure 9.

Alternatively, ρ-bromobenzaldehyde (5) can be converted to(d/l)-threo-p-bromophenylglycine (6, Scheme 1), (Bolhoffer, W. A. J. Am.Chem. Soc. 1954, 76, 1322; Herbert, R. B.; Wilkinson, B.; Ellames, G. J.Can. J. Chem. 1994, 72, 114), which can be protected as N-Boc derivative7 and then reduced in two-steps: activation with DCC andN-hydroxysuccinimide followed by treatment with NaBH₄ to provide 8.Compound 9 can be isolated as its TFA salt or as the free base.Regioselective introduction of fluoride is accomplished by protectingthe amine and benzylic hydroxyl group as phenyl oxazoline 10 followed byfluorination with (diethylamino)sulfur trifluoride (DAST) or[bis(2-methoxyethyl)amino]sulfur trifluoride (Lal, G. S., J. Org. Chem.,1999, 64, 7048) to give 11. Compound 11 is used in the Suzukicross-coupling reaction for the synthesis of biaryl derivatives. Theother Suzuki partner, an aryl boronic acid such as 12, can be preparedas shown and reacted with aryl halides.

The conditions for removal of the phenyloxazoline protecting groupproved to be incompatible with many functional groups at the p-positionof the aromatic ring. Thus, a new protecting group motif was developedbased on literature methods (Scheme 2; Jommi, G., et al., Gazz. Chim.Ital., 1986, 116:485). Phenylserine 6 was converted to methyl ester 13,which was protected as oxazolidinone 14, which, in turn, was reducedwith NaBH₄ to 15. Fluorination with either DAST or[bis(2-methoxyethyl)amino]sulfur trifluoride gave intermediate 16, whichdid not give consistently good Suzuki reaction yields. Thus, 16 wasconverted to 17. Cleavage of the oxazolidinone protecting group wasfacilitated by the introduction of a Boc group (Grehn, L., et al., ActaChem. Scand. B, 1986, 40, 745) followed by base-catalyzed cleavage togive 18 (Ishizuka, T. and Kunieda, T., Tetrahedron Lett., 1987, 28,4185; Jommi, G., et al., Gazz. Chim. Ital., 1988, 118:75).

Compound 18 was protected as isopropylidene derivative 19, which wasconverted to boronic acid 23 (Scheme 3). Compounds 18, 19, 22 and 23were used in Suzuki cross-coupling reactions. Compound 23 provided themost versatility since it could be cross-coupled with any aryl bromideor iodide and the protecting groups could be easily removed under mildconditions. Compound 22 was attractive as a Suzuki cross-couplingpartner in that the coupling reactions yielded the desired florfenicolanalogs directly without the need for deprotection anddihaloacetylation. However, cross-coupling yields with 22 were lowerthan those with 23. Cross-couplings using 21 gave the desired productscontaminated with acetate and monochloroacetate analogs.

Racemic compound 20 was identical spectroscopically to a samplesynthesized from semi-synthetic 11 by hydrolytic cleavage and basicwork-up. This provided unequivocal proof that the condensation ofphenylserine 6 had proceeded with the appropriate relative threo or synstereoselectivity.

Compounds 11 and 12 were used to prepare compounds 28 and 29 (Scheme 4).Compound 11 was cross-coupled with 3-pyridine boronic acid understandard biphasic Suzuki conditions to yield protected intermediate 26.The N—C cross-coupling product 27 was prepared according to the recentlyreported methods of Lam and coworkers (Lam, P. Y. S., et al., Synlett,2000, 674; Lam, P. Y. S., Tetrahedron, Lett., 1998, 39, 2941; Lam, P. Y.S., Tetrahedron Lett., 2001, 42, 3415). Both 26 and 27 were deprotectedand dichloroacetylated to give 28 and 29.

Compound 23 and 2-bromo-1,3,4-thiadiazole (24) were reacted to give 48(Scheme 5). Suzuki cross-coupling produced the heterobiaryl 25. Compound25 was deprotected with 9:1 TFA/H₂O, which removed the Boc andisopropylidene groups simultaneously, and then was difluoroacetyated togive 48.

i) Cs₂CO₃, PdCl₂(dppf), THF, DMF, H₂O; ii) 9:1 TFA/H₂O; iii) Et₃N,methyl difluoroacetate, Δ

While ρ-bromo functional groups of derivatives such as 11 and thecorresponding boronic acids such as 12 have substantial utility assynthetic intermediates to florfenicol analogs, ρ-cyano compounds havealso served as important intermediates. Using literature methods(Morris, D. S.; Smith, S. D., above), intermediates such as 30 have beenprepared in enantiomerically pure form (Scheme 6). Larger amounts ofintermediate were prepared using racemic, totally syntheticintermediates such as 35. The aldol condensation used to produce 32 fromρ-cyanobenzaldehyde and glycine methyl ester was adapted from theliterature (Pines, S. H. and Kazlowski, M. A., J. Org. Chem., 1972, 37,292).

Compound 41 (Scheme 7) was prepared from intermediate 36, thepreparation of which from 30 has been described (von Strandtmann, M., etal., J. Med. Chem., 1967, 10, 888). Compound 36 was deprotected withaqueous H₂SO₄ and then regioselectively protected as phenyloxazoline 38.Compound 39 was obtained on treatment with DAST and then was deprotectedand dichloroacetylated to give 41.

Compound 47 was prepared from intermediate 35 by treatment withhydroxylamine hydrochloride followed by triethyl orthoformate to give43, which was deprotected to give 46, which was then dichloroacetylated.

ρ-Acyl derivatives were obtained by Stille coupling reactions of theprotected intermediates (Scheme 9, compounds 87 and 88) with acidchlorides. Trimethylstannyl groups were introduced by Pd-mediatedreactions using hexamethylditin.

The cyclobutyl derivative 73 was prepared from 87 via a Stille couplingreaction that produced intermediate 89 (Scheme 10) which was deprotectedand dichloro-acetylated.

Attempted deprotection of the cyclopropyl derivative corresponding to 89led to HCl-mediated ring opening. Thus, to prepare 91, theboc/isopropylidene approach was employed since deprotection occurs underconditions that do not affect the cyclopropyl group.

When required, ρ-carboxyphenyl derivatives of protected phenicolintermediates could be prepared in two ways. For example, hydrolysis ofthe ρ-nitrile analog of 17 gives the corresponding carboxylic acid.However, nitrites generally could not be obtained as readily as thebromo derivatives. Thus, the preferred approach to carboxylation wasreplacement of the bromo group. For example, carboxylic acid derivative102 (Scheme 11) was prepared by lithiation of 17 followed by treatmentwith CO₂ and acid work-up. Compound 102 was converted to methyl ester103, which was reacted with hydrazine to give 104. Cyclization of 104with triethyl orthoformate gave oxadiazole 105, which was deprotectedand dihaloacetylated to give 106 and 107.

The carboxylic acid derivative of 102 was also used to prepare compoundssuch as 109 (Scheme 12), by reduction to alcohol 108 followed bydeprotection and dichloroacetylation

Biological Evaluation

All of the compounds of this invention are expected to demonstrateantimicrobial activity against the same bacteria as the other members ofthe chloramphenicol family. In addition, they may be expected to beactive against species of bacteria that are resistant to currentchloramphenicol antibiotics, in particular florfenicol. It is alsoexpected that the present compounds may exhibit activity against generaand species of bacteria against which current chloramphenicol-typeantibiotics are not active.

It is also understood that, with regard to bioactivity, one enantiomerof a compound may be more active than the other. In such case, whetherexpressly stated or not, the more active isomer is considered thepreferred embodiment of this invention. Particularly preferred is themost active enantiomer of the 1-(R)-2(S) absolute configuration of anycompound herein.

To determine the range and level of activity of the compounds of thisinvention, the following protocols may be used. Other such protocolswill become apparent to those skilled in the art based on thedisclosures herein and are within the scope of this invention. Somecompounds herein are expected to not only exhibit substantialantibacterial activity but to also be less susceptible to currentchloramphenicol resistance mechanisms. The screening protocols hereinmay be used to determine such characteristics also.

Susceptibility Testing

Compounds were evaluated against a panel of bacterial strains using abroth microdilution assay performed as recommended by the NCCLS(National Committee for Clinical Laboratory Standards (NCCLS) 2000,Methods for Dilution of Antimicrobial Susceptibility Tests for BacteriaThat Grow Aerobically—Fifth Edition, Approved Standard, NCCLS DocumentM7-A5, Vol 20, No. 2). The minimum inhibitory concentration (MIC) isdefined as the lowest concentration of a compound that prevents thegrowth of the bacteria.

The following 10 organisms constituted the primary panel of evaluation:TABLE 2 Efflux Bacteria Strain Pump Phenotype Escherichia coli ECM1194AcrAB^(a) Wild type Escherichia coli ECM1694 None ΔacrAB::Tn903kmrEscherichia coli ECM 1642 AcrAB MarR Escherichia coli ECM 1888 MdfAtolC::Tn mdfR Escherichia coli ECM 1750 CmlA ΔacrAB::Tn903kmr/ pLQ821Escherichia coli ECM 1958 Flo ΔacrAB::Tn903kmr/ p1956 Escherichia coliECM 1970 AcrAB + marR/p1956 Flo Escherichia coli ECM 1197 AcrAB^(a)EMR::Cm (cat) or ECM 2024 AcrAB^(a) ECM1197 acrAB::Kan PasteurellaATCC43134 Wild type multocida Mannhemia ATCC33396 Wild type haemolytica^(a)AcrAB efflux pump expressed at a low level

Assays were performed in Cation-Adjusted Mueller-Hinton Broth (CAMHB) ata bacterial inoculum of 5×10⁵ CFU/ml and a final volume of 100 μl.Florfenicol and chloramphenicol controls and test compounds wereprepared at four times the desired final concentration. Dilution to thedesired concentration was accomplished directly on the plates by serial2-fold dilution using a multi-channel pipette. After dilution, 25 μl ofCAMHB was added to each well.

The bacterial inocula were prepared as follows. For each strain, oneisolated colony was used to inoculate a volume of 5 ml of CAMHB. Thecultures were incubated overnight (20 hours) at 35° C. in a shakingincubator. They were then diluted in sterile saline to a densityequivalent to a 0.5 McFarland suspension (approx. 10⁸ CFU/ml). Thesuspensions were further diluted in CAMHB to approximately 5×10⁵ CFU/ml.A volume of 50 μl of the inoculum was added to each well. Positive andnegative growth controls were included on each plate. The originalinocula were determined by applying 10 μl of several 10-fold dilutionson TSA plates. Agar plates were incubated overnight at 35° C. andcolony-forming units (CFU) counted. Microtiter plates were incubated for20 hours at 35° C. and were read using a microtiterplate reader(Molecular Devices) at 650 nm and by visual observation using amicrotiterplate reading mirror to determine the MIC.

Pharmaceutical Compositions

A compound of the present invention, a prodrug thereof or aphysiologically acceptable salt of either the compound or its prodrug,can be administered as such to a patient or can be administered inpharmaceutical compositions in which the foregoing materials are mixedwith suitable excipient(s). Techniques for formulation andadministration of drugs may be found in Remington's PharmacologicalSciences, Mack Publishing Co., Easton, Pa., latest edition. Theformulations and techniques discussed in Remington relate primarily touse with human patients; however, they may readily modified for use withnon-human patients by techniques well-known to those skilled in theveterinary art.

Routes of Administration

As used herein, “administer” or “administration” refers to the deliveryof a compound, salt or prodrug of the present invention or of apharmaceutical composition containing a compound, salt or prodrug ofthis invention to an organism for the purpose of treating or preventinga microbial infection.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal, intramuscular, subcutaneous, intramedullary,intrathecal, direct intraventricular, intravenous, intravitreal,intraperitoneal, intranasal, aural or intraocular. The preferred routesof administration are oral and parenteral.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, by preparation as a salve that is applieddirectly to the infected area or by injection of the compound directlyinto infected tissue. In either case, a sustained release formulationmay be used.

Composition/Formulation

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., using a variety of well-knownmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. The compositionsmay be formulated in conjunction with one or more physiologicallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

For injection, including, without limitation, intravenous,intramusclular and subcutaneous injection, the compounds of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, physiological saline buffer or polar solvents including,without limitation, N-methyl-2-pyrrolidone, 2-pyrrolidone, otherpyrrolidones, N,N-dimethylacetamide, N,N-dimethylformamide,dimethylsulfoxide, acetone and glycerol formal. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carrierswell-known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, lozenges, dragees,capsules, liquids, gels, syrups, pastes, slurries, solutions,suspensions, concentrated solutions and suspensions for diluting in thedrinking water of a patient, premixes for dilution in the feed of apatient, and the like, for oral ingestion by a patient. Pharmaceuticalpreparations for oral use can be made using a solid excipient,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding other suitable auxiliaries if desired, to obtaintablets or dragee cores. Useful excipients are, in particular, fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol,cellulose preparations such as, for example, maize starch, wheat starch,rice starch and potato starch and other materials such as gelatin, gumtragacanth, methyl cellulose, hydroxypropyl-methylcellulose, sodiumcarboxy-methylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid. A salt such as sodium alginate mayalso be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.

For administration by inhalation, the compounds of the present inventioncan conveniently be delivered in the form of an aerosol spray using apressurized pack or a nebulizer and a suitable propellant, e.g., withoutlimitation, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be controlled by providing avalve to deliver a metered amount. Capsules and cartridges of, forexample, gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Useful compositions include, without limitation,suspensions, solutions or emulsions in oily or aqueous vehicles, and maycontain adjuncts such as suspending, stabilizing and/or dispersingagents. Pharmaceutical compositions for parenteral administrationinclude aqueous solutions of a water soluble form, such as, withoutlimitation, a salt, of the active compound. Additionally, suspensions ofthe active compounds may be prepared in a lipophilic vehicle. Suitablelipophilic vehicles include fatty oils such as sesame oil, syntheticfatty acid esters such as ethyl oleate and triglycerides, or materialssuch as liposomes. Aqueous injection suspensions may contain substancesthat increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers and/or agents thatincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Alternatively, the active ingredient maybe in powder form for constitution with a suitable vehicle, e.g.,sterile, pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

Other delivery systems for relatively hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well-knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, organic solvents such as dimethylsulfoxide may be used,although often at the risk of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semi-permeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the particularcompound, additional stabilization strategies may be employed.

Pharmaceutical compositions useful herein also may comprise solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Dosage

A therapeutically effective amount refers to an amount of compoundeffective to prevent, alleviate or ameliorate symptoms of a microbialinfection. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the disclosure herein.

For any compound used in the methods of the invention, thetherapeutically effective amount can be estimated initially from cellculture assays. Then, the dosage can be formulated for use in animalmodels so as to achieve a circulating concentration range that includesthe MIC as determined in cell culture. Such information can then be usedto more accurately determine dosages useful in patients.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals. For example, the MIC and the LD₅₀ for a particularcompound can be determined by methods well-known in the art. The dataobtained can be used to formulat a range of dosages useful in patients.The dosage, of course, may vary depending upon the dosage form and routeof administration. The exact formulation, route of administration anddosage can be selected by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1). In general,however, the presently preferred dosage range for systemic delivery of acompound of this invention will be from about 1 to about 100 mg/Kg. Thepresently preferred dosage range for topical use will generally be fromabout 0.1 mg to about 1 gm.

Dosage amount and interval may be adjusted individually to provideplasma levels of the compound that are sufficient to maintain aconcentration equal to the MIC or any other desired level. Such plasmalevels are often referred to as minimum effective concentrations (MECs).The MEC will vary for each compound but can be estimated from in vitrodata, e.g., the concentration necessary to achieve 80+% inhibition of amicrobe, may be ascertained using the assays described herein. Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the patient being treated, the severity of the infection, the mannerof administration, the judgment of the prescribing physician, etc.

Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

EXAMPLES

The following examples are provided to illustrate certain embodiments ofthis invention and are not intended, nor are they to be construed, tolimit its scope in any manner whatsoever.

Starting materials were obtained from commercial suppliers and usedwithout further purification unless otherwise noted. Chemical suppliersincluded Aldrich, Fluka, and Lancaster. Pd(PPh₃)₄ was obtained fromLancaster or Strem and immediately transferred into N₂ flushed vials(between 10 and 100 mg each) in an N₂ glove bag. The vials were wrappedin aluminum foil and stored in N₂-flushed, zip-lock baggies at −20° C.PdCl₂(dppf) was obtained from Aldrich and used from the bottle. Standardreagent grade solvents, which were not necessarily anhydrous, were used.Anhydrous solvents were purchased from chemical suppliers and used asis.

¹H NMR spectra were recorded on a 300 MHz Varian FT-NMR spectrometer andare reported in the format “chemical shift (multiplicity, integration,coupling constant).” Coupling constants are reported in Hz. Mass spectrawere obtained on a Micromass Platform II single quadrupole massspectrometer equipped with electrospray ionization (ESI).

The following three Suzuki cross-coupling methods were used:

Method A: the aryl boronic acid (0.167 mmol) and aryl bromide (0.334mmol) were combined in a mixture of aqueous Na₂CO₃ (3 mL of a 10% (w/w)solution) and THF (5 mL). The mixture was purged briefly with N₂.Pd(PPh₃)₄ (10 mol %, 0.0167 mmol) was added, the mixture purged with N₂,and then refluxed for 16 hours. The reaction mixture was diluted withethyl acetate (EtOAc), washed with a saturated brine solution, driedover anhydrous Na₂SO₄ and concentrated. The residue was then purified bychromatography.

Method B: the aryl boronic acid (0.301 mmol), the aryl bromide (0.602mmol), and Cs₂CO₃ (0.903 mmol) were combined in THF (2.0 mL), DMF (2,0mL), and H₂O (0.5 mL) at room temperature. The mixture was purged withN₂ for 5 min and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)dichloromethane complex (0.0301 mmol) was added. The mixture was purgedwith N₂ for 5 minutes and then stirred at 55° C. for 16 hours. Themixture was concentrated under vacuum, diluted with EtOAc and washedwith brine. The EtOAc was dried over anhydrous Na₂SO₄ and concentrated.The residue was purified by chromatography.

Method C: the aryl boronic acid (0.934 mmol), the aryl bromide (2.34mmol), and Cs₂CO₃ (2.80 mmol) were suspended in a mixture of toluene (4mL), n-butanol (4 mL), and H₂O (2 mL). The mixture was purged at roomtemperature with N₂ after which Pd(PPh₃)₄ (0.280 mmol) was added. Themixture was purged with N₂ for an additional 5 minutes and then heatedto 75° C. After 12 hours, the mixture was cooled to room temperature andconcentrated under vacuum. The residue was partitioned between H₂O (75mL) and EtOAc (75 mL). The EtOAc layer was washed with brine (2×50 mL),dried over anhydrous Na₂SO₄, and concentrated under vacuum. The residuewas purified by chromatography.

Example 1 Compound 3

In 80 mL of methanol was dissolved 8.13 g (32.0 mmol) of chloramphenicolbase as the N-acetate, prepared by literature methods (Rebstock, M. C.,et al., supra, Crooks H. M. et al., supra; Evans, D. D.; Morris, D. S.et al., supra). After purging with N₂, Pd/C was added and the mixturewas stirred under H₂(1 atm) for 16 hours. The Pd/C was then removed byfiltration through Celite. The solution was concentrated under vacuumand the residue purified by silica gel chromatography, eluting with 15%MeOH in CH₂Cl₂ to give 3 (7.13 g, 31.7 mmol). ¹H NMR (300 MHz, CD₃OD): δ1.92 (s, 3H), 3.40 (dd, 1H, J=11.1, 6.0), 3.61 (dd, 1H, J=1.1, 5.7),4.01 (m, 1H), 4.73 (d, 1H, J=5.4), 6.69 (d, 2H, J=8.4), 7.11 (d,2H,J=8.4).

Example 2 Compound 4

An aqueous solution of NaNO₂ (1.27 g, 18.5 mmol, 50 mL H₂O) was addeddropwise to a solution of 3 (3.76 g, 16.8 mmol) in 35 mL of 48% aqueousHBr at 0° C. After addition was completed, the mixture was stirred for30 min at 0° C. The mixture was then added dropwise to a solution ofCuBr (2.65 g, 18.45 mmol) in 15 mL 48% aqueous HBr. The mixture waswarmed to room temperature and stirred for an additional 16 hours. Thereaction mixture was neutralized with 3 M aqueous NaOH, filtered througha pad of Celite, and extracted with EtOAc (3×100 mL). The combinedorganic fractions were dried over anhydrous Na₂SO₄ and concentratedunder vacuum to give 1.2 g (4.1 mmol) of crude product that was usedwithout further purification. ¹H NMR (300 MHz, CD₃OD): δ 1.87 (s, 3H),3.48 (dd, 1H, J=11.0, 5.9), 3.69 (dd, 1H, J=11.0, 6.5), 4.01 (m, 1H),4.91 (d, 1H, J=3.6), 7.30 (d, 2H, J=8.3), 7.45 (d, 2H, J=8.3).

Example 3 Compound 6

4-Bromobenzaldehyde (100 g, 0.540 mol) was dissolved in ethanol (EtOH)in a 2 L round-bottom flask. With rapid stirring, glycine (0.5 molarequivalents, 20.3 g, 0.270 mol) and then, in one portion, KOH (30.3 g,0.540 mol) were added. It is essential to add the KOH all at once. Thus,an ice bath should be used when performing the reaction on a largerscale to control the exotherm. After addition of the KOH, the turbidsuspension became yellow and homogeneous. After about 15 minutes, athick white precipitate began to form. The mixture was stirred for 12hours at room temperature under N₂. Enough 2 N aqueous HCl was added(˜400 mL) to make the solution red to pH paper. The mixture was thenstirred at approximately 60° C. until it again become a homogeneousyellow solution. The EtOH was removed under vacuum to give an aqueoussuspension of white precipitate. The precipitate was filtered and theremaining aqueous solution washed three times with EtOAc. The aqueoussolution was then basified to about pH 9 with concentrated aqueous NH₃and excess ammonia was removed under vacuum. As the NH₃ was removed, theproduct, 6, began to precipitate. Evaporation continued to one-quarterthe volume of solution where precipitation was first observed. Theproduct was then collected on a vacuum filter and dried under vacuum toa constant weight (51.3 g). If NMR indicates the presence of theundesired trans stereoisomer, it can be removed by recrystallizationfrom H₂O/EtOH). ¹H NMR (300 MHz, CD₃OD): δ 3.64 (d, 1H, J=3.6), 5.25 (d,1H, J=3.6), 7.41 (d, 2H, J=8.4), 7.53 (d, 2H, J=8.4).

Example 4 Compound 7

Compound 6 (32.60 g, 12.53 mmol) was dissolved in 10% aqueous K₂CO₃ (10%w/v; 500 mL). Di-tert-butyl dicarbonate (34.2 g, 157 mmol) was dissolvedin 1,4-dioxane (500 mL) and added to the aqueous solution, after whichthe mixture was stirred at room temperature for 72 hours. The mixturewas concentrated under vacuum and taken up in 100 mL 1 N aqueous NaOHand washed with Et₂O (2×100 mL). The aqueous layer was acidified with 1N aqueous HCl and the product extracted into EtOAc (3×200 mL). Thecombined EtOAc extracts were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The product (26.7 g, 74.0 mmol) was isolatedand used without further purification. ¹H NMR (300 MHz, CD₃OD): δ 1.32(s, 9H), 4.37 (d, 1H, J=2.7), 5.23 (d, 1H, J=2.7), 7.32 (d, 2H, 8.4),7.46 (d, 2H, J=8.4).

Example 5 Compound 8

Compound 7 (5.29 g, 14.7 mmol) and N-hydroxysuccinimide (1.69 g, 14.7mol) were dissolved in EtOAc (200 mL) and the mixture cooled to 0° C.N,N′-dicyclohexyl-carbodiimide (3.04 g, 14.7 mmol) was added and themixture stirred for 30 minutes, warmed to room temperature and stirredfor 30 additional minutes. It was then cooled to 0° C. and filtered toremove the precipitated N,N′-dicyclohexylurea byproduct. The filtratewas concentrated under vacuum and the residue dissolved in THF (100 mL).The solution was cooled to 0° C., NaBH₄ (5.6 g, 150 mmol) added, and themixture stirred for two minutes. Water was then added to the mixturedropwise until bubbling ceased, then a volume of water equal to thevolume of THF was added over 30 minutes, after which the mixture waswarmed to room temperature and stirred for 5 hours. The dioxane wasremoved under vacuum and EtOAc (200 mL) was added, followed by gradualacidification of the aqueous layer with 1 N aqueous HCl. The EtOAc layerwas washed with brine and dried over anhydrous Na₂SO₄. Filtrationfollowed by concentration under vacuum gave 4.31 g (12.4 mmol) of 8,which was used without further purification. ¹H NMR (300 MHz, CD₃OD): δ1.32 (s, 9H), 3.46-3.51 (m, 1H), 3.63-3.73 (m, 2H), 4.88 (br s, 1H),7.29 (d, 2H, J=8.3), 7.45 (d, 2H, J=8.3).

Example 6 Compound 9 (From 4)

Compound 4 (1.0 g, 3.5 mmol) was dissolved in 10% aqueous H₂SO₄ (10%v/v, 15 mL total) and the solution refluxed for 10 h. The reactionmixture was basified with 3 M aqueous NaOH and extracted with EtOAc(3×50 mL). The combined EtOAc fractions were dried over anhydrous Na₂SO₄and concentrated under vacuum to give 710 mg (2.9 mmol) of 4, which wasused without further purification. ¹H NMR (300 MHz, CD₃OD): δ 2.80-2.90(br m, 1H), 3.30-3.39 (br m, 1H), 3.40-3.50 (br m,1H), 4.52-4.58 (m,1H), 7.30 (d, 2H, J=8.1), 7.49 (d, 2H, J=8.1).

Example 7 Compound 9 (from 8)

Compound 8 (4.31 g, 12.4 mmol) was dissolved in trifluoroacetic acid(TFA, 40 mL). The mixture was stirred for 3 hours at room temperatureand concentrated under vacuum. The TFA salt was partitioned between 50mL of 2 N aqueous NaOH and an equal volume of EtOAc. The layers wereseparated and the aqueous layer was washed with EtOAc (50 mL). Thecombined EtOAc fractions were washed with brine, dried over anhydrousNa₂SO₄ and concentrated under vacuum to give a waxy solid (2.72 g, 11.1mmol), which was used without further purification. ¹H NMR (300 MHz,CD₃OD): δ 2.83-2.90 (m, 1H), 3.30-3.35 (m, 1H), 3.46 (dd, 1H J=10.8,4.8), 4.56 (d, 1H, J=6.6), 7.29 (d, 2H, J=8.6), 7.49 (d, 2H, J=8.6).

Example 8 Compound 10

Compound 9 (710 mg, 2.89 mmol), ethyl benzimidate hydrochloride (533 mg,2.89 mmol) and triethylamine (Et₃N, 0.40 mL, 2.89 mmol) were combined in25 mL 1,2-dichloroethane. The mixture was refluxed with stirring underN₂ for 16 hours at which time TLC indicated one major product. Aftercooling to room temperature, the mixture was diluted with EtOAc, washedtwice with saturated aqueous NH₄Cl, twice with saturated aqueous NaHCO₃and then dried over anhydrous Na₂SO₄. The solution was filtered and theEtOAc removed under vacuum to give 885 mg (2.66 mmol) of 10, which wasused without further purification. ¹H NMR (300 MHz, CD₃OD): δ 3.74-3.89(m, 2H), 4.13-4.18 (m, 1H), 5.59 (d, 1H, J=6.6), 7.30 (d, 2H, J=8.4),7.46-7.57 (m, 5H), 7.99-8.02 (m, 2H).

Example 9 Compound 11

Compound 10 (885 mg, 2.66 mmol) was suspended in CH₂Cl₂ (15 mL) andcooled to −78° C. Diethylaminosulfur trifluoride (DAST, 0.53 mL, 4.00mmol) was added by syringe, the mixture warmed over 1 hour to roomtemperature and then stirred for 16 hours under N₂. Excess DAST wasquenched by slow addition of H₂O after which the mixture was dilutedwith additional CH₂Cl₂. The organic layer was washed with H₂O andsaturated aqueous NaHCO₃, dried over anhydrous Na₂SO₄ and concentratedunder vacuum. The residue (950 mg) was chromatographed on silica gel,eluting with 15:85 EtOAc/hexanes to give 11 as an oil (420 mg, 1.25mmol). ¹H NMR (300 MHz, CDCl₃): δ 4.23-4.35 (m, 1H), 4.45-4.75 (m, 2H),5.43 (d, 1H, J=6.9), 7.17(d, 2H, 8.3), 7.36-7.47 (m, 5H), 7.96 (d, 2H,J=8.3).

Example 10 Compound 12

Compound 11 (807 mg, 2.41 mmol, enantiopure from chloramphenicol orracemic from (±)-threo-p-bromophenylserine), was dissolved in anhydrousTHF (10 mL) in a flame-dried round bottom flask and cooled to −78° C.n-BuLi (1.6 M in hexanes, 3.02 mL, 4.83 mmol) was added with vigorousstirring. After 10 minutes, trimethyl borate (0.55 mL, 4.83 mmol) wasadded, the mixture warmed to room temperature over 1 hour and thenstirred for 6 hours. The mixture was quenched with 1 N aqueous HCl andextracted three times with EtOAc. The combined EtOAc fractions werewashed three times with brine and dried over anhydrous Na₂SO₄. The EtOAcwas removed under vacuum to give 805 mg of material, which was purifiedby flash column chromatography, eluting initially with 1:1 hexanes/EtOActo remove impurities followed by elution with 1:9 MeOH/CH₂Cl₂ to obtainthe product as an oil (285 mg, 0.953 mmol). ¹H NMR (300 MHz, CD₃OD): δ4.29-4.39 (m, 1H), 4.69 (dd, 2H, J=47.1, 4.2), 5.63 (d, 1H, J=6.6), 7.37(d, 2H, J=8.1), 7.47-7.52 (m, 2H), 7.56-7.62 (m, 1H), 7.67 (d, 1H,J=7.5) 7.74-7.82 (m, 1H), 8.01 (d, 2H, J=7.2).). LRMS (ESI⁻) m/z: 298.0(M−H⁺C₁₆H₁₄BFNO₃ requires 298.1).

Example 11 Compound 13

Compound 6 (20.6 g, 0.079 mol) was suspended in 400 mL of anhydrousMeOH. The mixture was stirred rapidly and cooled in an ice bath to 0° C.Anhydrous HCl gas was slowly bubbled in. After 10 minutes, all solidshad dissolved. Acidification was continued for 10 minutes after themixture became homogeneous, at which point the MeOH turned pH paper veryred. The apparatus was fitted with a drying tube and the reactionmixture was refluxed for 8 hours followed by stirring at roomtemperature for 12 hours. The mixture was concentrated under vacuum andthen suspended in a mixture of 300 mL of EtOAC and 100 mL of water. Withrapid stirring, 3 N NaOH was added very slowly until the suspendedmaterial dissolved. Addition of base was continued until a pH of 10 wasattained. The layers were separated and the EtOAc fraction was washedwith brine (2×) and dried over anhydrous Na₂SO₄. Concentration undervacuum gave 13 as a white powder (15.3 g), which was used withoutfurther purification. ¹H NMR (300 MHz, CD₃OD): δ 3.59 (d, 1H, J=4.5),3.66 (s, 3H), 4.92 (d, 1H, J=4.5), 7.29 (d, 2H, J=8.3), 7.49 (d, 2H,J=8.3).

Example 12 Compound 14

1,1′-Carbonyldiimidazole (23.6 g, 0.146 mol) was dissolved in 250 mL ofanhydrous 1,2-dichloroethane at room temperature followed by addition oftriethylamine (Et₃N, 12.2 g, 16.8 mL). In a separate flask, 13 (33.26 g,0.121 mol) was dissolved in 125 mL of anhydrous tetrahydrofuran (THF)and an equivalent of triethylamine. The THF solution was diluted with125 mL of 1,2-dichloroethane and then added, under N₂, over 2 hours tothe 1,1′-carbonyldiimidazole solution. After addition was complete, themixture was stirred for 2 hours under N₂ at room temperature. Thereaction mixture was concentrated under vacuum and the residue dilutedwith 300 mL of EtOAc, which was washed with 5×200 mL of 2 N HCl, 3×300mL of saturated NaHCO₃, and 2×200 mL of brine. The EtOAc layer was driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give34.27 g of 14, 80-90% pure by ¹H NMR. Compound 14 was used withoutfurther purification. ¹H NMR(300 MHz, CD₃OD): δ 3.83 (s, 3H), 4.34 (d,1H, J=5.0), 5.65 (d, 1H, J=5.0), 7.36 (d, 2H, J=8.7), 7.60 (d, 2H,J=8.7).

Example 13 Compound 15

Compound 14 (55.3 g, 0.184 mmol) was dissolved in 375 mL MeOH. Thesolution was cooled to 0° C. and NaBH₄ (2 equivalents, 13.9 g, 0.369mol) was added in portions, care being taken to not let the temperatureexceed 20° C. After the last portion was added, cooling was ceased andthe mixture stirred for 2 hours. Glacial acetic acid was added slowlyuntil a pH of 7 was achieved. The mixture was filtered through Celiteand concentrated under vacuum. The residue was partitioned between EtOAc(375 mL) and 2 N HCl (500 mL). The organic layer was washed with 2 N HCl(2×300 mL), saturated aqueous NaHCO₃ (2×250 mL) and brine (250 mL),dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum. Theproduct was crystallized from hexanes/EtOAc to give 26.3 g of 15. ¹HNMR(300 MHz, CD3OD): δ 3.64-3.72 (m, 3H), 5.38 (d, 1H, J=5.1), 7.32 (d,2H, J=8.6), 7.57 (d, 2H, J=8.6).

Example 14 Compound 16

Compound 15 (7.57 g, 0.0278 mol) was suspended in 275 mL of CH₂Cl₂.Under N₂ and with rapid stirring, the biphasic mixture was cooled to−78° C. in a dry ice/acetone bath. DAST (5.51 mL, 6.72 g, 0.0417 mol)was added dropwise by syringe over 2-3 minutes. After 30 minutes themixture was transferred to an ice water bath and stirred for 30 minutes,during which time the solids dissolved. Another 1 mL of DAST was addedto ensure completion of the reaction. The mixture was stirred for 20minutes at 0° C. and then warmed to room temperature. The mixture wasstirred at room temperature for 20 minutes and then cooled to 0° C.Excess DAST was quenched by dropwise addition of saturated aqueousNaHCO₃ over 30 minutes with very rapid stirring. After bubbling ceased,the aqueous layer was slightly basic (7<pH<9). The CH₂Cl₂ was dilutedwith enough EtOAc (˜600 mL) to bring the organic layer to the top duringaqueous extraction. The organic layer was washed with saturated aqueousNaHCO₃ (1×300 mL), 1 N HCl (2×200 mL), saturated aqueous NaHCO₃ (3×200mL) and brine (1×150 mL) and dried over anhydrous Na₂SO₄. Afterfiltration, the mixture was concentrated under vacuum to give 8.13 g ofbrown oil. ¹H NMR(300 MHz, CD₃OD): δ 3.87-3.98 (dm, 1H, J=20(F—CH_(N))), 4.54 (dd, 2H, J=46.7 (CH₂—F), 4.2), 5.42 (d, 1H, J=5.1),7.34 (d, 2H, J=8.4), 7.59 (d, 2H, J=8.4).

Example 15 Compound 17

Compound 16 (8.13 g or 0.0297) was dissolved in 200 mL of CH₃CN. Boc₂O(9.71 g, 1.5 equivalents, 0.0445 mol) and DMAP (362 mg, 0.00297 mol, 0.1equivalent) were then added. The mixture was stirred for 2 hours at roomtemperature under N₂. Concentration of the mixture under vacuum wasfollowed by partitioning between 200 mL of EtOAc and 200 mL of 1 N HCl.The EtOAc layer was washed with 2 N HCl (3×100 mL), saturated aqueousNaHCO₃ (2×100 mL) and brine. The EtOAc layer was filtered, dried overanhydrous Na₂SO₄ and concentrated under vacuum. The residue wasrecrystallized twice from hexanes/EtOAc. ¹H NMR (300 MHz, CD₃OD): δ 1.53(s, 9H), 4.29-4.40 (dm, 1H, J=24.6), 4.63-4.98 (m, 2H), 5.53 (d, 1H,J=4.2), 7.34 (d, 2H, J=8.4), 7.61, (d, 2H, J=8.4).

Example 16 Compound 18

Compound 17 (0.050 g, 0.134 mmol) was suspended in 5 mL of CH₃OH. Cs₂CO₃(8.7 mg, 20 mol %, 0.0267 mmol) was added with rapid stirring at roomtemperature. The solids dissolved in about 10 minutes after whichstirring was continued for 10 minutes, at which time TLC (3:1hexanes/EtOAc) indicated that the reaction was complete. The reactionmixture was concentrated under vacuum and the residue partitionedbetween 20 mL of EtOAc and 10 mL of H₂O. The H₂O layer was acidifiedslightly with 1N aqueous HCl, the mixture vigorously shaken and thelayers separated. The EtOAc layer was washed with saturated aqueousNaHCO₃ (1×20 mL) and brine (1×20 mL). The EtOAc was dried over anhydrousNa₂SO₄ and concentrated under vacuum to give 43.4 mg (0.124 mmol) of 18as a white foam. ¹H NMR (300 MHz, CD₃OD): δ 1.33 (s, 9H), 3.90-4.57 (m,4H), 7.29 (d, 2H, J=8.1), 7.46 (d, 2H, J=8.1)

Example 17 Compound 22

Compound 18 (0.740 g, 2.13 mmol) was stirred in 10 mL TFA/H₂O (9/1, v/v)at room temperature for one hour. The mixture was concentrated undervacuum and partitioned between 30 mL of EtOAc and 30 mL of 1N aqueousNaOH. The mixture was shaken vigorously and the layers allowed toseparate. The organic layer was washed with brine (2×), dried overanhydrous Na₂SO₄ and concentrated under vacuum to give 0.484 g (1.95mmol, 92%) of product, which was dissolved in 20 mL of CH₂Cl₂. To thiswas added 1 mL of Et₃N and 1 mL of difluoroacetyl chloride, preparedaccording to literature procedure (Yu, K.-L., et al., J. Med. Chem.,1996, 39, 2411-2421.). After 1 hour, the mixture was concentrated undervacuum. The residue was placed in 10 mL of a 1:8:1 (v/v/v) solution ofEt₃N/MeOH/H₂O and stirred for one hour at room temperature to cleave anydifluoroacetyl groups on the benzylic oxygen. The mixture was thenconcentrated under vacuum, and partitioned between EtOAc (50 mL) and 1 NHCl (50 ml). The EtOAc layer was washed twice with 20 mL of 1 N HCl,twice with 20 mL saturated aqueous NaHCO₃ and twice with 20 mL brine.The EtOAc was then dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. Compound 22 (584 mg, 1.79 mmol) was usedwithout further purification. ¹H NMR (300 MHz, CD₃OD): δ 4.25-4.67 (m,3H), 4.90 (d, 1H, J=3.9), 5.98 (t, 1H, J 53.9), 7.30 (d, 2H, J=8.6),7.48 (d, 2H, J=8.6).

Example 18 Compound 19

Compound 18 (1.03 g, 2.94 mmol) was dissolved in CH₂Cl₂ (30 mL) in a 50mL round-bottom flask. 2-Methoxypropene (339 μL, 3.54 mmol) was added bysyringe, followed by a single crystal of ρ-toluenesulfonic acid (ρ-TsOH)hydrate. After about 1 minute, the mixture turned yellow. TLC (4:1hexanes/EtOAc) showed some 18 to still be present. An additional 5 dropsof 2-methoxypropene were added followed two minutes later by another 5drops, a which time TLC indicated the reaction was complete. The CH₂Cl₂was removed under vacuum and the residue partitioned between EtOAc (75mL) and saturated aqueous NaHCO₃ (50 mL). The EtOAc layer was washedtwice with 50 mL NaHCO₃ and twice with brine. The EtOAc layer was driedover anhydrous Na₂SO₄, filtered and evaporated to give a yellow oil(1.08 g, 2.78 mmol) that solidified to a waxy solid, which was usedwithout further purification. ¹H NMR (300 MHz, CD₃OD): δ 1.49 (s, 9H),1.55 (s, 3H), 1.67 (s, 3H), 3.70-3.85 (m, 1H), 4.34-4.53 (m, 2H), 5.08,(d, 1H, J=7.5), 7.37 (d, 2H, J=8.6), 7.54 (d, 2H, J=8.6).

Example 19 Compound 23

Compound 19 (309 mg, 0.796 mmol) was placed in an oven-dried 50 mLround-bottom flask equipped with a magnetic stir bar. The flask wasimmediately capped with a rubber septum and flushed with dry N₂.Anhydrous THF (15 mL) was added by syringe and a long cannula was usedto flush the solvent with N₂ for 10 minutes. The mixture was then cooledto −78° C. With vigorous stirring, n-BuLi (850 μL, 0.995 mmol) was addeddropwise by syringe over 3 minutes to give a clear orange solution.After 20 minutes, B(OMe)₃ (158 μL, 1.39 mmol), which had been storedover 4 Å activated molecular sieves for at least 48 hours, was addeddropwise over one minute using an oven-dried, gas-tight syringe. Themixture was warmed to room temperature and stirred under N₂for 16 hours.Approximately 20 mL of saturated aqueous NH₄Cl was added with vigorousstirring, which resulted in a white precipitate dispersed in the twoliquid phases. The mixture was stirred for an additional 90 minutes andthen diluted with EtOAc and H₂O until all the precipitate dissolved. Theorganic layer was separated, washed with brine and dried over anhydrousNa₂SO₄. After filtration, the EtOAc was removed under vacuum to give 328mg of yellow oil. The product was purified by silica gel chromatographyto give 23 (163 mg). ¹H NMR (300 MHz, CD₃OD): δ 1.49 (s, 9H), 1.56 (s,3H), 1.67 (s, 3H), 3.74-3.88 (m, 1H), 4.33-1.52 (m, 2H), 5.10 (d, 1H,J=7.5), 7.43 (d, 2H, J=8.1), 7.64 (d, 2H, J=8.1). LRMS (ESI⁻) m/z: 352.2(M−H⁺ C₁₇H₂₄BFNO₅ requires 352.7).

Example 20 Compound 28

This is an example of Suzuki coupling method A. Compound 11 (47 mg, 0.14mmol) and pyridine-3-boronic acid (21 mg, 0.17 mmol) were dissolved in5.0 mL of THF. Pd(PPh₃)₄ (11 mg, 0.0098 mmol) and 3.0 mL of 10% (w/v)aqueous Na₂CO₃ were added. The reaction mixture was refluxed for 16hours, cooled, diluted with EtOAc and washed twice with 10% (w/v)aqueous Na₂CO₃ and twice with brine. The EtOAc layer was then dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. Silica gelchromatography (eluting with 1:1 hexanes/EtOAc) gave 47 mg (0.14 mmol)of 26. Intermediate 26 was heated to 105° C. in a sealed tube with 6Maqueous HCl and held at that temperature for 16 hours. The mixture wascooled to room temperature and basified to pH 10 with 3 N NaOH. Theproduct was extracted into EtOAc, which was dried over Na₂SO₄, filtered,and concentrated under vacuum to give 42 mg of the deprotected freeamine, contaminated with the expected benzoic acid-related by-productarising from cleavage of the protecting group. The crude residue wasdichloroacetylated as in the synthesis of 29. Silica gel chromatography,eluting with 7.5% MeOH in CH₂Cl₂, gave 10 mg of product. ¹H NMR (300MHz, CD₃OD): δ 4.31-4.74(m, 3H), 5.02 (d, 1H, J=3.6), 6.27 (s, 1H),7.49-7.56 (m, 3H), 7.64 (d, 2H, J=8.4), 8.09 (ddd, 1H, J=8.0, 2.3, 1.5),8.50 (br d, 1H, J=3.9), 8.78 (br s, 1H). LRMS (ESI⁺) m/z: 356.9 (calc.for M+H⁺: C₁₆H₁₆Cl₂FN₂O₂ 357.0).

Example 21 Compound 27

Boronic acid 12 (43 mg, 0.144 mmol), imidazole (15 mg, 0.215 mmol) and 4Å powdered molecular sieves (110 mg) were combined in CH₂Cl₂ (4.0 mL)and pyridine (23 μL). With rapid stirring, Cu(OAc)₂ (26 mg, 0.144 mmol)was added and the mixture stirred, exposed to air, for 40 hours at roomtemperature. The mixture was quenched with 3 mL of 2 M NH₃ in MeOH. Themixture was filtered through Celite and concentrated under vacuum. Theproduct was purified by flash column chromatography (19:1 CH₂Cl₂/MeOH).Although ¹H NMR indicated that a significant side-product had elutedwith the desired product, the mixture was used without furtherpurification. LRMS (ESI⁺) m/z: 321.9 (calc. for M+H⁺ C₁₉H₁₇FN₃O 322.1).

Example 22 Compound 29

Compound 27 (27 mg, 0.084 mmol) was heated with 6 N HCl (3.0 mL) to 100°C. in a sealed tube and held for 16 hours. The mixture was cooled toroom temperature, basified with 3 N aqueous NaOH and extracted threetimes with EtOAc. The combined EtOAc fractions were dried over anhydrousNa₂SO₄, filtered, and concentrated under vacuum to give 9 mg of residue.LRMS (ESI⁺) m/z: 236.1 (Calc. for M+H⁺ C₁₂H₁₅FN₃O 236.1).

The residue was refluxed in MeOH (5.0 mL) containing Et₃N (26 μL 0.189mmol) and methyl dichloroacetate (13 μL, 0.126 mmol) for 3 hours. Themixture was concentrated under vacuum and purified by silica gelchromatography (7.5% MeOH in CH₂Cl₂) to give 29 (6.0 mg, 0.017 mmol). ¹HNMR (300 MHz, CD₃OD): δ 4.33-4.75 (m, 3H), 5.03 (d, 1H, J=3.3), 6.26 (s,1H), 7.13 (s, 1H), 7.51-7.58 (m, 5H), 8.10 (s, 1H). LRMS (ESI⁺) m/z:345.8 (calc. for M+H⁺ C₁₄H₁₅Cl₂FN₃O₂ 346.0).

Example 23 Compound 34

Compound 32 was prepared by the procedure of Pines, et al., supra. In a250-mL round-bottom flask was placed 1,1′-carbonyldiimidazole (CDI, 5.06g, 31.2 mmol) and Et₃N (2.17 mL, 15.6 mmol) in 1,2-dichloroethane (50mL). Compound 32 (4.00 g, 15.6 mmol) was added to a beaker containing1,2-dichloroethane (50 mL). Et₃N (4.34 mL, 31.2 mmol) was added to thebeaker upon which a thick suspension formed. Additional1,2-dichloroethane (20 mL) and THF (50 mL) were added to thin thesuspension. The mixture was added portion-wise to the CDI suspensionover 30 min. The mixture, which became mostly homogenous and turnedyellow was stirred for 12 hours. The solvent was removed under vacuumand the residue was partitioned between 2 N aqueous HCl and EtOAc. TheEtOAc layer was washed with 2 N HCl (2×45 mL), saturated aqueous NaHCO₃(2×30 mL) and brine. It was dried over anhydrous Na₂SO₄, filtered, andevaporated to give 3.27 g of 33 as a fluffy yellow solid, which was usedwithout further purification.

Compound 33 (1.00 g,4.06 mmol) was dissolved in MeOH (50 mL) and cooledto 0° C. in an ice bath. Over 5 minutes, NaBH₄ (308 mg, 8.13 mmol) wasadded portion-wise. After bubbling ceased, TLC (5% MeOH in CH₂Cl₂)indicated that starting material remained so 100 mg additional NaBH₄ wasadded. After bubbling ceased, the mixture was quenched with glacial HOAcuntil a pH of approximately 7 was achieved. The mixture was concentratedunder vacuum and partitioned between EtOAc and 1 N HCl. The EtOAc layerwas washed with saturated aqueous NacHCO₃ (2×40 mL) and brine (40 mL)and dried over anhydrous Na₂SO₄. The product was isolated by silica gelchromatography (2% to 3% to 4% to 10% MeOH in CH₂Cl₂) to give 128 mg of34 as an oil. Additional product was obtained by evaporation of theaqueous iayer followed by trituration with hot EtOAc (153 mg, 281 mgtotal). ¹H NMR (300 MHz, CD₃OD): δ 3.69 (br s, 3H), 5.50 (br s, 1H),7.58 (d, 2H, J=7.8), 7.79 (d, 2H, J=7.8).

Example 24 Compound 35

Fluorination was performed using DAST as in the synthesis of compound11. ¹H NMR (300 MHz, CD₃OD): δ 3.89-3.97 (m, 1H), 4.49-4.66 (m, 2 H),5.55 (d, 1H, J=4.8), 7.59 (d, 2H, J=8.1), 7.80 (d, 2H, J=8.4).

Example 25 Compound 38

Compound 36 (2.12 g), was prepared by the procedure of von Strandtmann,supra. It was heated with 10% aqueous H₂SO₄ to 100° C. in a sealed tubeand held for 4 hours. After cooling to room temperature, the mixture wasbasified with 1 M NaOH and extracted three times with n-butanol. Then-butanol fractions were combined and dried over anhydrous Na2SO₄. Thesolvent was evaporated to give 1.61 g of 37.

Compound 37 (1.61 g, 7.70 mmol) was dissolved in 1,2-dichloroethane (100mL), along with ethyl benzimidate hydrochloride (1.42 g, 7.70 mmol), andEt₃N (1.06 mL, 7.70 mmol). The mixture was refluxed for 12 hours, cooledto room temperature and diluted with EtOAc. The solution was washed withsaturated aqueous NH₄Cl (3×) and saturated NaHCO₃ (1×) and dried overanhydrous Na₂SO₄. The product precipitated from the EtOAc on cooling andaddition of several drops of hexanes to give 0.62 g of 38 (2.1 mmol). ¹HNMR (300 MHz, CD₃OD): δ 2.60 (s, 3H), 3.79 (dd, 1H, J=11.3, 5.9), 3.89(dd, 1H, J=11.3, 4.1), 4.14-4.20 (m, 1H), 5.70 (d, 1H, 6.3), 7.47-7.61(m, 5H), 8.01-8.05 (m, 4H).

Example 26 Compound 39

Compound 38 (0.62 g, 2.1 mmol) was suspended in CH₂Cl₂ (15 mL) andcooled to −78° C. DAST (0.42 mL, 3.1 mmol) was added by syringe and thesolution stirred overnight, during which time it was allowed to come toroom temperature as the cold bath warmed. The solvent was removed undervacuum and the product isolated by silica gel chromatography (1:4EtOAc/hexanes) to give 0.17 g of 39. ¹H NMR (300 MHz, CD₃OD): δ 2.60 (s,3H), 4.30-4.40 (m, 1H), 4.63-4.67 (m, 1H), 4.79-4.81 (m, 1H), 5.74 (d,1H, 6.9), 7.48-7.63 (m, 5H), 8.01-8.07 (m, 4H).

Example 27 Compound 40

Compound 39 (0.17 g, 0.58 mmol) was suspended in 6 N aqueous HCl (5 mL)in a sealed tube. The mixture was heated to 100° C. and held for 12hours. After cooling to room temperature, the mixture was basified with3 N NaOH and the product extracted into CH₂Cl₂ (3×). The combined CH₂Cl₂fractions were dried over anhydrous Na₂SO₄, filtered and concentrated togive 40 (0.113 g, 0.541 mmol). ¹H NMR (300 MHz, CD₃OD): δ 2.60 (s, 3H),3.04-3.15 (m, 1H), 4.09-4.48 (m, 3H), 4.71 (d, 1H, J=6.0), 7.52 (d, 2H,J=8.1), 7.90 (d, 2H, J=8.1).

Example 28 Compound 41

Compound 40 was dichloroacetylated to give 41 in the same manner that 27was converted to 29. ¹H NMR (300 MHz, CD₃OD): δ 2.57 (s, 3H), 4.31-4.78(m, 3H,), 5.03 (d, 1H, J=3.3), 6.23 (s, 1H), 7.53 (d, 2H, J=8.6), 7.95(d, 2H, J=8.6).

Example 29 Compound 43

Compound 35 (100 mg, 0.454 mmol) was dissolved in 3 mL of EtOH andtransferred to a 25 mL round-bottom flask. Hydroxylamine hydrochloride(38 mg, 0.545 mmol) was added followed by Et₃N (127 μL, 0.909 mmol). Themixture was stirred at reflux for 3 hours, at which point TLC indicatedcomplete consumption of starting material. The mixture was concentratedunder vacuum to 232 mg of yellow oil, which was used without furtherpurification. A portion of the oil (115 mg) was dissolved in 10 mL oftriethylorthoformate. The mixture was stirred at 120° C. under N₂ for 2hours and then at room temperature for 48 hours. TLC (10% MeOH inCH₂Cl₂) a major new spot. The triethyl orthoformate was removed undervacuum and the residue partitioned between EtOAc and 1 N aqueous NaOH.The organic layer was washed with 1 N aqueous NaOH (2×) and brine (2×).The EtOAc layer was dried over anhydrous Na₂SO₄, filtered, andevaporated, to give 84 mg of oil. The product was purified bychromatotron (1 mm silica plate, 60:40 to 50:50 to 40:60 hexanes/EtOAc).Another purification by chromatotron (2% MeOH in CH₂Cl₂) gave 43 (20 mg)as a white solid. ¹H NMR (300 MHz, CD₃OD): δ 3.93-4.04 (m, 1H),4.49-4.67 (m, 2 H), 5.53 (d, 1H, J=5.1), 7.59 (d, 2H, J=8.3), 8.17 (d,2H, J=8.3), 9.28 (s, 1H).

Example 30 Compound 45

Compound 43 (20 mg, 0.076 mmol) was dissolved in CH₃CN (2 mL). Boc₂O (25mg, 0.114 mmol) was added followed by a single crystal of DMAP(approximately 1 mg, 0.008 mmol). After 2 hours, TLC (1:1 hexanes/EtOAc)indicated that the reaction was complete. The CH₃CN was removed undervacuum and the resulting white solid dissolved in EtOAc, which waswashed with 1 N aqueous HCl (2×), followed by NaHCO₃ (2×) then brine.The organic layer was dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum to give 44 as a white solid (28 mg).

Compound 44 (0.026 mg, 0.072 mmol) was dissolved in MeOH (2 mL) andCs₂CO₃ (5 mg, 0.014 mmol) was added in a single portion. After 1 hour,TLC (1:1 hexanes/EtOAc) showed a single product. The solvent was removedunder vacuum, EtOAc added and the mixture stirred rapidly at roomtemperature. Then, H₂O was added, followed by 0.5 M HCl dropwise untilall solids dissolved. The organic phase was washed with 0.5 N HCl,followed by saturated aqueous NaHCO₃ (2×) and brine. The organic layerwas then dried over anhydrous Na₂SO₄, filtered and concentrated to givea yellow oil, which was purified by chromatotron (1 mm silica plate, 4:1hexanes/EtOAc to 1:1 hexanes/EtOAc to give 45 (13 mg, 0.039 mmol). ¹HNMR (300 MHz, CD₃OD): δ 1.31 (s, 9H), 4.01-4.63 (m, 3 H), 4.94 (d, 1H,J=2.7), 7.55 (d, 2H, J=8.4), 8.05 (d, 2H, J=8.4), 9.24 (s, 1H).

Example 31 Compound 47

Compound 45 was dissolved in 2.5 mL of 9:1 TFA/H₂O in a 10 mL flask. Themixture was stirred at room temperature for 30 minutes, at which timeTLC indicated completion of the reaction. The mixture was concentratedunder vacuum and partitioned with rapid stirring between 1 N aqueousNaOH and EtOAc. The EtOAc layer was washed with brine (1×), dried overanhydrous Na₂SO₄, filtered and evaporated to give 11 mg of 46 as an oil.

To 6 mg (0.025 mmol) of 46 in a 10 mL round bottom flask was added MeOH(2 mL), followed by Et₃N (3.5 μL, 0.025 mmol) and methyl dichloroacetate(13.1 μL, 0.025 mmol). The mixture was refluxed for 4 hours at whichtime TLC indicated no reaction had occurred. An additional 26.2 μL ofmethyl dichloroacetate was added followed by 7 μL of Et₃N. The mixturewas refluxed for 20 hours after which TLC indicated that the reactionwas complete. The product was purified via chromatotron (1 mm silica gelplate, 99:1 CH₂Cl₂/MeOH) to give 5 mg 47. ¹H NMR (300 MHz, CD₃OD): δ4.32-4.72 (m, 3H), 5.03 (d, 1H, J=2.7), 6.25 (s, 1H), 7.56 (d, 2H,J=8.3), 8.05 (d, 2H, J=8.3), 9.24 (s, 1H). LRMS (ESI⁻) m/z: 346.0 (M−H⁺C₁₃H₁₁Cl₂FN₃O₃ requires 346.0).

Example 32 Compound 24

In a 200 mL round-bottom flask, 2-amino-1,3,4-thiadiazole (1.00 g, 9.89mmol) was added to 10 mL of 48% aqueous HBr. Water (10 mL) was added togive a yellow solution in which most solids dissolved. The mixture wascooled to 0° C. and CuBr (142 mg, 0.989 mmol) was added to give anopaque brown solution with some precipitate. NaNO₂ (682 mg, 9.89 mmol)was dissolved in 25 mL H₂O and added dropwise over 45 minutes to thethiadiazole mixture. The mixture became dark green and opaque with thefirst drops of NaNO₂ solution. Slowly, the solution became brownishyellow and brown gas evolved. The mixture was stirred for an additional10 minutes at 0° C. from the time gas evolution began. The mixture waswarmed to room temperature over 30 minutes. Saturated aqueous NaHCO₃ wasadded dropwise until bubbling ceased and the pH was 8.5. To this wasadded 50 mL of EtOAc and the biphasic mixture stirred rapidly. Themixture was filtered through a pad of Celite to remove solids and thenthe layers were separated. The organics were washed with brine (2×). Theaqueous layer was extracted with EtOAc and washed with brine (1×). Thecombined organic layers were dried over Na₂SO₄. The remaining aqueousmaterial was extracted once more by vigorous stirring with EtOAc (50 mL)for 12 hours. This EtOAc layer was washed with brine and combined withthe EtOAc that was already drying over Na₂SO₄. The solution was filteredand evaporated under vacuum to give 24 as a tan solid (1.22 g, 7.36mmol), which was used without further purification.

Example 33 Compound 25

This is an example of Suzuki coupling method B. Compound 23 (19 mg,0.0538 mmol) was dissolved in THF (1 mL), DMF (1 mL), and H₂O (0.5 mL).The mixture was stirred at room temperature until all material dissolvedand then 2-bromo-1,3,4-thiadiazole (24, 5.0 mg, 0.027 mmol) was added.The solution was purged with N₂ for 5 minutes by means of a long needle.PdCl₂(dppf) was added and then the solution was again purged with N₂ for5 minutes. The mixture was then stirred at 55° C. for 18 hours under N₂and then at room temperature for an additional 30 hours. The resultingclear brown solution was concentrated under vacuum. Compound 25 (5.4 mg,0.0137 mmol) was obtained as a yellow oil by chromatotron purification(silica gel, 1 mm plate, 1:9 EtOAc/hexanes to 1:4 EtOAc/hexanes).

It was found that the yield could be substantially improved by switchingto coupling method C. Compounds 23 (0.330 g, 0.934 mmol) and 24 (0.385g, 2.34 mmol) were dissolved in a mixture of toluene, n-butanol, and H₂O(8 mL:8 mL:2 mL) and Cs₂CO₃ (0.912 g, 2.80 mmol) was added. The mixturewas purged with N₂ and Pd(PPh₃)₄ was added. The mixture was again purgedwith N₂ for 5 minutes and then stirred rapidly at 70° C. under N₂ for 12hours. The mixture was concentrated under vacuum and the residuepartitioned between H₂O (75 mL) and EtOAc (75 mL. The layers wereseparated and the EtOAc was washed with brine (2×50 mL). The EtOAc wasdried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum togive 0.720 g of yellow oil. Compound 25 (0.220 g) was obtained by silicagel chromatography, eluting with 5:1 to 2:1 to 1:1 hexanes/EtOAc. ¹H NMR(300 MHz, CD₃OD): δ 1.50 (s, 9H), 1.58 (s, 3H), 1.70 (s, 3H), 3.80-3.94(m, 1 H), 4.42-4.61 (m, 2H), 5.20, (d, 1H, J=7.5), 7.64 (d, 2H, J=8.3),8.05 (d, 2H, J=8.3), 9.45 (s, 1H).

Example 34 Compound 48

Compound 25 (5.4 mg, 0.014 mmol) was transferred to a 25 mL round-bottomflask and 2.5 mL of 9:1 TFA/H₂O (v/v) were added. The resulting brightyellow mixture was stirred at room temperature for 18 hours. The solventwas removed under vacuum and the residue dissolved three times in amixture of MeOH and toluene, which was evaporated to near dryness eachtime. The product was recovered as an oil (4.9 mg, 0.013 mmol), whichwas used without further purification. ¹H NMR (300 MHz, CD₃OD): δ3.59-3.70 (m, 1H), 4.30-4.71 (m, 2H), 4.92 (d, 1H, J=8.1), 7.64 (d, 2H,J=8.3), 8.08 (d, 2H, J=8.3), 9.47 (s, 1H).

In a 10 mL round-bottom flask, the above product was dissolved in 2 mLof MeOH. To this was added Et₃N (9.7 μL, 0.070 mmol) followed by methyldifluoroacetate (3.0 μL, 0.035 mmol). The mixture was refluxed for 16hours, at which point TLC indicated that some starting material stillremained. An additional 3 drops of Et₃N was added followed by 2 drops ofmethyl difluoroacetate. After 3 hours, all starting material had beenconsumed. The mixture was cooled to room temperature and evaporated todryness under vacuum. Compound 48 (4.1 mg, 012 mmol) was obtained bychromatotron purification (silica gel, 1 mm plate). ¹H NMR (300 MHz,CD₃OD): δ 4.31-4.72 (m, 3H), 5.03 (d, 1H, J=3.9), 5.98 (t, 1H, J=53.9),7.58 (d, 2H, J=8.3), 7.99 (d, 2H, J=8.3), 9.43 (s, 1H). LRMS (ESI⁻) m/z:330.1 (M−H⁺ C₁₃H₁₁F₃N₃O₂S requires 330.1).

Example 35 Compound 49

The biaryl intermediate was prepared from 18 and the appropriate boronicacid using Suzuki coupling method A. Removal of the Boc group wasaccomplished by brief treatment with 90/10 (v/v) TFA/H₂O.Dichloroacetylation was performed as in the synthesis of 29. ¹H NMR (300MHz, CD₃OD): δ 4.31-4.80 (m, 3H), 5.01 (d, 1H, J=3.6), 6.26 (s, 1H),7.52 (d, 2H, 8.3), 7.65 (d, 2H, J=8.3), 7.79 (s, 4H). LRMS (ESI⁻) m/z:379.0 (M−H⁺ C₁₈H₁₄C₁₂FN₂O₂ 379.0).

Example 36 Compound 50

The biaryl intermediate was prepared from 22 and the appropriate boronicacid using Suzuki coupling method A. ¹H NMR (300 MHz, CD₃OD): δ4.27-4.68 (m, 3H), 4.99 (d, 1H, J=4.5), 6.00 (t, 1H, J=53.9), 7.52 (d,2H, J=8.1), 7.67 (d, 2H, J=8.1), 7.76-7.82 (m, 2H). LRMS (ESI⁻) m/z:347.1 (M−H⁺ C₁₈H₁₄F₃N₂O₂ requires 347.1).

Example 37 Compound 51

The biaryl intermediate was prepared from 22 and the appropriate boronicacid using Suzuki coupling method A. ¹H NMR (300 MHz, CD₃OD): δ 3.14 (s,3H), 4.30-4.68 (m, 3H), 5.00 (d, 1H, J=4.5), 6.00 (t, 1H, J=54), 7.53(d, 2H, J=8.3), 7.70 (d, 2H, J=8.3), 7.88 (d, 2H, J=8.6), 8.00 (d, 2H,J=8.6). LRMS (ESI⁻) m/z: 400.1 (M−H⁺ C₁₈H₁₇F₃NO₄S requires 400.1).

Example 38 Compound 52

The biaryl intermediate was prepared from 22 and the appropriate boronicacid using Suzuki coupling method B. ¹H NMR (300 MHz, CD₃OD): δ4.26-4.64 (m, 3H), 4.93 (d, 1H, J=4.8), 6.01 (t, 1H, J=53.9), 7.40-7.45(m, 4H), 7.60-7.66 (m, 3H). LRMS (ESI⁻) m/z: 328.1 (M−H⁺ C15H₁₃F₃NO₂Srequires 328.1).

Example 39 Compound 53

The biaryl intermediate was prepared from 22 and the appropriate boronicacid using Suzuki coupling method B. ¹H NMR (300 MHz, CD₃OD): δ4.25-4.65 (m, 3H), 4.92 (d, 1H, J=4.5), 6.01 (t, 1H, J=53.9), 7.06-7.08(m, 1H), 7.36-7.42 (m, 4H), 7.61 (d, 2H, J=8.4). LRMS (ESI⁺) m/z: 352.0(calc. for M+Na⁺ C₁₅H₁₄F₃NNaO₂S 352.1).

Example 40 Compound 54

The biaryl intermediate was prepared from 18 and the boronic acid usingSuzuki coupling method B. Removal of the Boc group was accomplished bybrief treatment with 9/1 (v/v) TFA/H₂O. Dichloroacetylation wasperformed as in the synthesis of 29. ¹H NMR (300 MHz, CD₃OD): δ4.33-4.79 (m, 3H), 5.03 (d, 1H, J=3.3), 6.26 (s, 1H), 7.55 (d, 2H,J=8.4), 7.69-7.75 (m, 4H), 8.56 (d, 2H, J=5.7). LRMS (ESI⁺) m/z: 357.1(calc. for M+H⁺ C₁₆H₁₆Cl₂FN₂O₂ 357.1).

Example 41 Compound 55

The biaryl intermediate was prepared from boronic acid 23 and compound24 using Suzuki coupling method B. Removal of the protecting groups wasaccomplished by brief treatment with 9/1 (v/v) TFANH₂O.Dichloroacetylation was performed as in the synthesis of 29. ¹H NMR (300MHz, CD₃OD): δ 4.34-4.76 (m, 3H), 5.04 (d, 1H, J=3.0), 6.24 (s, 1H),7.58 (d, 2H, J=8.3), 7.97 (d, 2H, J=8.3), 9.42 (s, 1H). LRMS (ESI⁻) m/z:362.0 (calc. for M−H⁺ C₁₃H₁₁Cl₂FN₃O₂S 362.0).

Example 42 Compound 56

Compound 54 (29.4 mg, 0.0824 mmol) was dissolved in 5 mL of CH₂Cl₂. Themixture was cooled to 0° C. and m-chloroperbenzoic acid (m-CPBA, 38 mg,0.16 mmol) was added with stirring. After 5 minutes, the mixture waswarmed to room temperature and stirred for 12 hours. The mixture wasconcentrated under vacuum and the product purified by silica gelchromatography eluting successively with 2%, 3%, 4%, 6%, and 10% MeOH inCH₂Cl₂. Compound 56 was obtained as a white solid (22.5 mg, 0.060 mmol).¹H NMR (300 MHz, CD₃OD): δ 4.32-4.75 (m, 3H), 5.03 (d, 1H, J=3.3), 6.25(s, 1H), 7.56 (d, 2H, J=8.4), 7.74 (d, 2H, J=8.4), 7.84 (d, 2H, J=7.2),8.34 (d, 2H, J=7.2). LRMS (ESI⁻) m/z: 371.0 (calc. for M−H⁺C₁₆H₁₄Cl₂FN₂O₃ 371.0).

Example 43 Compound 57

The biaryl intermediate was prepared from 18 and the appropriate boronicacid using Suzuki coupling method B. Removal of the protecting group wasaccomplished by brief treatment with 9/1 (v/v) TFA/H₂O.Difluoroacetylation was performed as in the synthesis of 48. ¹H NMR (300MHz, CD₃OD): δ 4.28-4.68 (m, 3H), 5.00 (d, 1H, J=4.2), 5.99 (t, 1H,J=53.9), 7.55 (d, 2H, J=8.1), 7.70-7.77 (m, 4H), 7.84 (d, 2H, J=7.2),8.56 (d, 2H, J=6.3). LRMS (ESI⁺) m/z: 325.2 (calc. for M+H⁺ C₁₆H₁₆F₃N₂O₂325.1).

Example 44 Compound 58

Compound 58 was prepared from 68 as 56 was from 54. ¹H NMR (300 MHz,CD₃OD): δ 4.30-4.75 (m, 3H), 5.02 (d, 1H, J=3.3), 6.25 (s, 1H),7.55-7.67 (m, 5H), 7.87 (d, 1H, J=8.1), 8.30 (d, 1H, J=7.2), 8.58 (br s,1H). LRMS (ESI⁻) m/z: 371.0 (calc. for M−H⁺ C₁₆H₁₄Cl₂FN₂O₃ 371.0).

Example 45 Compound 59

The biaryl intermediate was prepared from 11 and the appropriate boronicacid using Suzuki coupling method A. The phenyloxazoline protectinggroup was removed and the dichloroacetate group introduced as in thesynthesis of 29. ¹H NMR (300 MHz, CD₃OD): δ 4.30-4.75 (m, 3H), 5.02 (d,1H, J=3.6), 6.26 (s, 1H), 7.54-8.70 (m, 8H). LRMS (ESI⁻) m/z: 354.8(calc. for M−H⁺ C₁₆H₁₄Cl₂FN₂O₂ 355.0).

Example 46 Compound 60

Compound 60 was prepared from compound 59 in the same manner that otherpyridine N-oxides in these examples were formed from the correspondingpyridine. ¹H NMR (300 MHz, CD₃OD): δ 4.33-4.78 (m, 3H), 5.03 (d, 1H,J=3.3), 6.25 (s, 1H), 7.57 (d, 2H, J=8.4), 7.75 (d, 2H, J=8.4), 7.87 (d,2H, J=7.5), 8.38 (d, 2H, J=7.5). LRMS (ESI⁻) m/z: 370.8 (calc. for M−H⁺C₁₆H₁₄Cl₂FN₂O₃ 371.0).

Example 47 Compound 61

The biaryl intermediate was prepared from 11 and the appropriate boronicacid using Suzuki coupling method A. The phenyl oxazoline protectinggroup was cleaved and dichloroacetate introduced as in the synthesis of29. ¹H NMR (300 MHz, CD₃OD): δ 3.14 (s, 3H), 4.36-4.75 (m, 3H), 5.02 (d,1H, J=3.6), 6.27 (s, 1H), 7.54 (d, 2H, J=8.3), 7.68 (d, 2H, J=8.3), 7.87(d, 2H, J=8.7), 8.01 (d, 2H, J=8.7). LRMS (ESI⁻) m/z: 431.8 (calc. forM−H⁺ C₁₈H₁₇Cl₂FNO₄S 432.0).

Example 48 Compound 62

The free amine intermediate was prepared as in the synthesis of 28. Thenitrogen was monochloroacetylated using chloroacetyl chloride. ¹H NMR(300 MHz, CD₃OD): δ 4.00-4.03 (m, 2H), 4.27-4.70 (m, 3H), 5.00 (d, 1H,J=3.9), 7.49-7.55 (m, 3H), 7.66 (d, 2H, J=8.4), 8.09 (d app t, 1H,J=7.8, 2.4), 8.50 (dd, 1H, J=4.8, 1.5), 8.78 (br d, 1H, J=2.4). LRMS(ESI⁺) m/z: 321.0 (calc. for M+H⁺ C₁₆H₁₅CIFN₂O₂ 357.1).

Example 49 Compound 63

Prepared in the same manner as 28 using ethyl chlorofluoroacetate. ¹HNMR (300 MHz, CD₃OD): δ 4.31-4.70 (m, 3H), 5.01 (d, 1H, J=3.9), majordiastereomer 6.49 (d, 1H, J=49.8), minor diastereomer 6.51 (d, 1H,J=49.8), 7.49-7.56 (m, 3H), 7.63-7.67 (m, 2H), 8.07-8.11 (m, 1H),8.49-8.51 (m, 1H), 8.78 (br d, 1H, J=1.5). LRMS (ESI) m/z: 338.9 (calc.for M−H⁺ C₁₆H₁₄CIF₂N₂O₂ 339.1).

Example 50 Compound 64

The biaryl intermediate was prepared from 12 and 2-bromopyridine usingSuzuki coupling method A. ¹H NMR (300 MHz, CD₃OD): δ 4.28-4.74 (m, 3H),5.02 (d, 1H, J=3.6), 6.27 (s, 1H), 7.33-7.37 (m, 1H), 7.53 (d, 2H,J=8.1), 7.18-7.93 (m, 4H), 8.58-8.60 (m, 1H). LRMS (ESI⁻) m/z: 354.8(calc. for M−H⁺ C₁₆H₁₄Cl₂FN₂O₂ 355.0)

Example 51 Compound 65

Compound 65 was prepared in the same manner as 59 using ethylchlorofluoroacetate. ¹H NMR (300 MHz, CD₃OD): δ 4.32-4.70 (m, 3H), 5.02(d, 1H, J=3.6), major diastereomer 6.48 (d, 1H, J=49.8), minordiastereomer 6.50 (d, 1H, J=50.1), 7.55-7.58 (m, 2H), 7.73-7.78 (m, 4H)8.57 (br d, 2H, J=5.7). LRMS (ESI⁻) m/z: 338.8 (calc. for M−H⁺C₁₆H₁₄CIF₂N₂O₂ 339.1).

Example 52 Compound 66

The biaryl intermediate was prepared from 12 and the appropriate bromideusing Suzuki coupling method A. Removal of the protecting group anddichloroacetylation was accomplished as in the synthesis of 29. ¹H NMR(300 MHz, CD₃OD): δ 4.34-4.72 (m, 3H), 5.01 (d, 1 H, J=2.4), 6.25 (s,1H), 7.52 (d, 2H, J=6.3), 7.57 (d, 1H, J=2.6) 7.84 (d, 1H, J=2.6), 7.90(d, 2H, J=6.3). LRMS (ESI⁻) m/z: 360.7 (calc. for M−H⁺ C₁₄H₁₂Cl₂FN₂O₂S361.0).

Example 53 Compound 67

The biaryl intermediate was prepared from 12 and the appropriate bromideusing Suzuki coupling method A. Removal of the protecting group anddichloroacetylation was accomplished as in the synthesis of 29. ¹H NMR(300 MHz, CD₃OD): δ 4.32-4.75 (m, 3H), 5.04 (d, 1H, J=3.6), 6.26 (s,1H), 7.59 (d, 2H, J=8.3), 7.70 (d, 2H, J=8.3) 9.06 (s, 2H,), 9.13 (s,1H). LRMS (ESI⁺) m/z: 357.8 (calc. for M+H⁺ C₁₅H₁₅Cl₂FN₃O₂ 358.0).

Example 54 Compound 68

The biaryl intermediate was prepared from bromide 11 and the appropriateboronic acid using Suzuki coupling method A. Removal of the protectinggroup and dichloroacetylation was accomplished as in the synthesis of29. ¹H NMR (300 MHz, CD₃OD): δ 4.31-4.74 (m, 3H), 5.02 (d, 1H, J=3.6),6.27 (s, 1H), 7.48-7.51 (m, 1H), 7.54 (d, 2H, J=8.3), 7.64 (d, 2H,J=8.3) 8.08 (ddd, 1H, J=8.0, 2.4, 1.8), 8.50 (dd, 1H, 4.8, 1.5), 8.77(dd, 1H, J=2.4, 0.9). LRMS (ESI⁻) m/z: 354.8 (calc. for M−H⁺C₁₆H₁₄Cl₂FN₂O₂ 355.0).

Example 55 Compound 69

The biaryl intermediate was prepared from bromide 11 and the appropriateboronic acid using Suzuki coupling method A. Removal of the protectinggroup and dichloroacetylation was performed as in the synthesis of 29.¹H NMR (300 MHz, CD₃OD): δ 2.63 (s, 3H), 4.31-4.74 (m, 3H), 5.01 (d, 1H,J=3.9), 6.27 (s, 1H), 7.51 (d, 2H, J=8.3), 7.67 (d, 2H, J=8.3), 7.75 (d,2H, J=8.6) 8.06 (d, 2H, J=8.6).

Example 56 Compound 71

This compound was prepared in analogous fashion to 41 using ethylchlorofluoroacetate in place of methyl dichloroacetate. ¹H NMR (300 MHz,CD₃OD): δ 2.58 (s, 3H), 4.31-4.71 (m, 3H), 5.03 (br d, 1H, J=3.9), majordiastereomer 6.46 (d, 1H, J=49.8), minor diastereomer (d, 1H, J=50.1),major diastereomer 7.53 (d, 2H, J=8.1), minor diastereomer 7.51 (d, 2H,J=8.1), 7.94-7.96 (m, 2H). LRMS (ESI⁻) m/z: 304.1 (calc. for M−H⁺C₁₃H₁₃CIF₂NO₃ 304.1).

Example 57 Compound 72

Initially, the ρ-cyano analog of compound 11 had been prepared bymethods analogous to those used to prepare 11 itself. Attempts to removethe phenyloxazoline protecting group (as described in the synthesis of29) led to some of the desired deprotected nitrile intermediate.However, much of the mass recovered was the deprotected ρ-carboxylicacid corresponding to acidic hydrolysis of the nitrile functional group.This material was dichloroacetylated using the same procedure as thatemployed in the synthesis of 29 and the product was converted to itsmethyl ester with CH₂N₂. ^(1 NMR ()300 MHz, CD₃OD): δ 3.83 (s, 3H),4.25-4.66 (m, 3H), 4.98 (d, 1H, J=3), 6.17 (s, 1H), 7.46 (d, 2H, J=8.3),7.91 (d, 2H, J=8.3).

Example 58 Compound 73

Compound 87 (36.5 mg, 0.0873 mmol) was dissolved in THF (5 mL anhydrous)under N₂. After addition of anhydrous powdered K₂CO₃ (24 mg, 0.175mmol), the mixture was purged with N₂. Et₃N (31 mL, 0.218 mmol) andcyclobutanecarbonyl chloride (13 mL, 0.113 mmol) were added and themixture purged gently with N₂ for another 5 min. Pd₂dba₃ was added andthe mixture purged again with N₂. The mixture was then stirred under N₂for 3 hours after which it was diluted with EtOAc and H₂O and filteredthrough a cotton plug. The EtOAc layer was washed with 1N aqueous HCl(×2), brine (×2), and dried over anhydrous Na₂SO₄. The mixture wasfiltered and the solvent removed to give 16.9 mg, 0.0501 mmol ofmaterial that was purified by chromatotron (1 mm plate, eluting with 4:1hexanes/EtOAc). The protected intermediate obtained was subjected tophenyloxazoline cleavage and dichloroacetylation as in the synthesis of29 to give 73. ¹H NMR (300 MHz, CD₃OD): δ 1.73-1.85 (m, 1H), 1.99-2.11(m, 1H), 2.20-2.27 (m, 4H), 3.97-4.09 (m, 1H), 4.23-4.67 (m, 3H), 4.95(d, 1H, J=3.3), 6.16 (s, 1H), 7.45 (d, 2H, J=8.4), 7.81 (d, 2H, J=8.4).LRMS (ESI⁻) m/z: 360.0 (calc. for M−H⁺ C₁₆H₁₇Cl₂FNO₃ 360.0).

Example 59 Compound 74

The carboxylic acid intermediate from the synthesis of 72 wasdichloroacetylated as in the synthesis of 29. The resulting intermediate(95.8 mg, 0.297 mmol) was dissolved in 2 mL of MeOH, several drops ofH₂O were added and then a 20% (w/v) aqueous Cs₂CO₃ solution was addeddropwise until the solution reached pH 7. The mixture was concentratedunder vacuum. One drop of Et₃N and 10 mL of 2-chloroethanol were addedand the mixture stirred at 135° C. for 2 hours. Residual chloroethanolwas removed under vacuum and the residue chromatographed. Compound 74(13.3 mg) was the major product and 75 (9.2 mg) the minor product. ¹HNMR (300 MHz, CD₃OD): δ 3.78-3.82 (m, 2H), 4.23-4.68 (m, 5H), 4.97 (d,1H, J=3.0), 6.17 (s, 1H), 7.46 (d, 2H, J=8.3), 7.93 (d, 2H, J=8.3). LRMS(ESI⁺) m/z: 407.9 (calc. for M+Na⁺ C₁₄H₁₅Cl₃FNNaO₄ 408.0).

Example 60 Compound 75

¹H NMR (300 MHz, CD₃OD): δ 3.83-3.39 (m, 2H), 4.31-4.74 (m, 5H), 5.02(d, 1H, J=3.3), 6.23 (s, 1H), 7.52 (d, 2H, J=8.4), 8.02 (d, 2H, J=8.4).LRMS (ESI³¹ ) m/z: 366.0 (calc. for M−H⁺ C₁₄H₁₅Cl₂FNO₅ 366.0).

Example 61 Compound 76

The cyclopropyl analog of intermediate 89 was prepared by a Stillecoupling analogous to that used to synthesize 89. Acidic cleavage of thephenyloxazoline group resulted in the HCl-mediated opening of thecyclopropyl ring. This material was deprotected and dichloroacetylatedto give 76. ¹H NMR (300 MHz, CD₃OD): δ 2.10-2.19 (m, 2H), 3.19 (t, 2H,J=7.1), 3.66 (t, 2H, J=6.6), 4.31-4.74 (m, 3H), 5.03 (d, 1H, J=3.3),6.23 (s, 1H), 7.53 (d, 2H, J=8.3), 7.96 (d, J=8.3). LRMS (ESI⁻) m/z:381.9 (calc. for M−H⁺ C₁₅H₁₆Cl₃FNO₃ 382.0).

Example 62 Compound 77

The thiazole ring of this analog was formed by conversion of theprotected nitrile intermediate to the corresponding thioamide byreaction with H₂S in pyridine. The thioamide was then reacted with theappropriate protected α-halocarbonyl compound to form the substitutedthiazole shown. Deprotection and dichloroacetylation was performed asfor other compounds of this invention. ¹H NMR (300 MHz, CD₃OD): δ4.32-4.75 (m, 5H), 5.01 (d, 1H, J=3.3), 6.27 (s, 1H), 7.38 (s, 1H), 7.51(d, 2H, J=8.3), 7.91 (d, 2H, J=8.3). LRMS (ESI⁻) m/z: 390.9 (calc. forM−H⁺ C₁₅H₁₄Cl₂FN₂O₃S 391.0).

Example 63 Compound 79

This compound was formed by Suzuki coupling method A from anintermediate in which the primary hydroxyl group was not converted to afluoride. Deprotection and dichloroacetylation were performed as withother compounds of this invention. ¹H NMR (300 MHz, CD₃OD): δ 3.55 (dd,1H, J=11.0, 5.9), 3.77 (dd, 1H, J=11.0, 6.5), 4.07-4.11 (m, 1H), 5.02(d, 1H, J=3.6), 5.96 (s, 2H), 6.29 (s, 1H), 6.86 (d, 1H, J=8.4),7.05-7.07 (m, 2H), 7.40-7.50 (m, 4H). LRMS (ESI⁻) m/z: 395.9 (calc. forM−H⁺ C₁₈H₁₆Cl₂NO₅ 396.0).

Example 64 Compound 80

Compound 41 (11.5 mg, 0.0346 mmol) and hydroxylamine hydrochloride (2.8mg, 0.0415 mmol) were stirred in EtOH (3 mL) for 12 hours and thenrefluxed for an additional 12 hours. The solvent was removed undervacuum and the product isolated as a single isomer (it was notdetermined whether the isomer was the cis or trans oxime). ¹H NMR (300MHz, CD₃OD): δ 2.21 (s, 3H), 4.26-4.68 (m, 3H,), 4.95 (d, 1H, J=3.9),6.27 (s, 1H), 7.39 (d, 2H, J=8.3), 7.61 (d, 2H, J=8.3).

Example 65 Compound 81

Free amine 37 (4.7 mg, 0.0224 mmol) was dissolved in MeOH (1 mL) and themixture cooled to 0° C. Chloroacetic anhydride (5 drops) andtriethylamine (5 drops) were added and the mixture warmed to roomtemperature and stirred for 2 hours. The solvent was removed undervacuum and the product purified by silica gel chromatography (2:3EtOAc/hexanes) to give 3.3 mg (0.011 mmol) of product. ¹H NMR (300 MHz,CD₃OD): δ 2.59 (s, 3H), 3.92-4.03 (m, 2H,), 4.29-4.68 (m, 3H), 5.02 (d,1H, J=3.3), 7.53 (d, 2H, J=8.4), 7.96 (d, 2H, J=8.4).

Example 66 Compound 82

The biaryl intermediate was formed by Suzuki coupling method A using 22and the appropriate boronic acid. ¹H NMR (300 MHz, CD₃OD): δ 4.28-4.68(m, 3H), 5.00 (d, 1H, J=4.2), 6.01 (t, 1H, J=54), 7.49-7.60 (m, 3H),7.66 (d, 2H, J=8.4), 8.07-8.11 (m, 1H), 8.50 (dd, 1H, J=4.8, 1.5),8.79-8.79 (m, 1H). LRMS (ESI⁻) m/z: 322.9 (M−H⁺ C₁₆H₁₄F₃N₂O₂ requires323.1).

Example 67 Compound 84

This compound was prepared by procedures analogous to those described inSchemes 2 and 3. The 4-(1,2,4-triazol-1-yl)phenylserine analog of 6 wassynthesized by the same methods used to make 6. The required4-(1,2,4-triazol-1-yl)benzaldehyde was prepared as described in theliterature (Tanaka, A., et al., J. Med. Chem.1998; 41, 2390-2410). ¹HNMR (300 MHz, CD₃OD): δ 4.33-4.76 (m, 3H), 5.04 (d, 1H, J=3.3), 6.26 (s,1H), 7.59 (d, 2H, J=8.6), 7.78 (d, 2H, J=8.6), 8.14 (s, 1H), 9.05 (s,1H). LRMS (ESI⁻) m/z: 345.0 (calc. for M−H⁺ C₁₃H₁₂Cl₂FN₄O₂ 345.0).

Example 68 Compound 85

The biaryl intermediate was prepared from boronic acid 12 and2-iodopyrazine. Deprotection and dichloroacetylation were accomplishedas in the synthesis of 29. ¹H NMR (300 MHz, CD₃OD): δ 4.32-4.75 (m, 3H),5.04 (d, 1H, J=3.3), 6.27 (s, 1H), 7.57 (d, 2H, J=8.4), 8.05 (d, 2H,J=8.4), 8.51 (d, 1H, J=2.7), 8.65-8.66 (m, 1H), 9.08 (d, 1H, J=1.8).LRMS (ESI⁻) m/z: 355.8 (calc. for M−H⁺ C₁₅H₁₃Cl₂FN₃O₂ 356.0).

Example 69 Compound 86

Same procedure as that for the synthesis of 68. ¹H NMR (300 MHz, CD₃OD):δ 4.29-4.73 (m, 3H), 5.01 (br d, 1H, J=3.9), major diastereomer 6.49 (d,1H, J=49.8), minor diastereomer 6.51 (d, 1H, J=49.8), 7.48-7.56 (m, 3H),7.64-7.67 (m, 2H), 8.06-8.10 (m, 1H), 8.49-8.51 (m, 1H), 8.78-8.79 (m,1H). LRMS (ESI⁻) m/z: 338.9 (calc. for M−H⁺ C₁₆H₁₄CIF₂N₂O₂ 339.1).

Example 70 Compound 87

Compound 11 (0.496 g, 1.48 mmol) was dissolved in anhydrous benzene (15mL) under N₂. The flask was fitted with a reflux condenser and a longneedle was dropped through the condenser and the solution purged with agentle stream of dry N₂ for 5 minutes. Hexamethylditin (0.58 g, 1.78mmol) was added and the mixture purged for another 5 minutes. Pd(PPh₃)₄(0.171 g, 0.148 mmol) was added and purging with N₂ continued. After 5minutes, the needle was removed and a N₂ inlet was placed at the top ofthe reflux condenser. The mixture was refluxed for approximately 2 hoursunder N₂. Over the course of the reaction, the mixture turned fromorange to yellow to black. The solvent was removed under vacuum and theproduct purified by chromatotron (silica gel, 1 mm plate) to give 440 mgof product as a clear oil. ¹H NMR (300 MHz, CD₃OD): δ 0.27 (s, 9H—thisresonance contains significant satellite peaks, which are due toNMR-active isotopes of tin), 4.27-4.41 (m, 1H), 4.59-4.76 (m, 2H), 5.60(d, 1H, J=6.9), 7.33 (d, 2H, J=8.1), 7.45-7.31 (m, 5H), 7.99-8.02 (m,2H).

Example 71 Compound 88

This compound was prepared in the same manner as 87. ¹H NMR (300 MHz,CD₃OD): δ 0.027 (s, 9H, with significant satellite peaks caused byNMR-active tin isotopes), 1.49 (s, 9H), 1.55 (s, 3H), 1.67 (s, 3H),3.73-3.83 (m, 1H), 4.30-4.49 (m, 2H), 5.06 (d, 2H, J=7.5), 7.36-7.51 (m,4H).

Example 72 Compound 89

Compound 87 (36.5 mg, 0.0873 mmol) was dissolved in anhydrous THF underN₂. With stirring, K₂CO₃ (24 mg, 0.16 mmol) was added. The mixture waspurged with a gentle stream of N₂ for 5 minutes. Et₃N (31 μL, 0.22 mmol)and cyclobutanecarbonyl chloride (13 μL, 0.11 mmol) were added and themixture purged with N₂ for 5 minutes. Pd₂dba₃ was added, the mixturepurged for 5 min and then stirred under N₂ for 3 hours. The mixture wasdiluted with EtOAc (25 mL) and H₂O (25 mL) and filtered through a cottonplug to remove solids. The EtOAc was washed with 1 N HCl (2×) and brine(2×), dried over Na₂SO₄ and concentrated under vacuum. The product waspurified by chromatotron (silica plate, 1 mm, 1:4 EtOAc/hexanes) to give16.9 mg (0.050 mmol) of product. LRMS (ESI⁺) m/z: 338.2 (M+H⁺ C₂₁H₂₁FNO₂requires 338.2).

Example 73 Compound 90

Same procedure as in the synthesis of 89. ¹H NMR (300 MHz, CD₃OD): δ0.027 (s, 9H, with significant satellite peaks caused by NMR-active tinisotopes), 1.49 (s, 9H), 1.55 (s, 3H), 1.67 (s, 3H), 3.73-3.83 (m, 1H),4.30-4.49 (m, 2H), 5.06 (d, 2H, J=7.5), 7.36-7.51 (m, 4H).

Example 74 Compound 91

Compound 91 was deprotected by brief treatment of 90 with 9/1 TFA/H₂O.The dichloroacetyl group was introduced as in the synthesis of 29. LRMS(ESI⁻) m/z: 345.8 (calc. for M−H⁺ C₁₅H₁₆Cl₂FNO₃ 346.0).

Example 75 Compound 92

The same procedure as that used in the synthesis of 56 was employed. ¹HNMR (300 MHz, CD₃OD): δ 4.28-4.74 (m, 3H), 5.01 (d, 1H, J=3.9), 5.98 (t,1H, J=53.8), 7.55 (d, 2H, J=8.4). 7.76 (d, 2H, J=8.4), 7.86 (d, 2H,J=7.1), 8.35 (d, 2H, J=7.1).

Example 76 Compound 93

The biaryl intermediate was prepared by reaction of 23 and theappropriate bromide using Suzuki coupling A. Deprotection anddichloroacetylation were carried out as described above in the synthesisof 29. LRMS (ESI⁻) m/z: 433.0 (M−H⁺ C₁₇H₁₆Cl₂FN₂O₄S requires 433.0).

Example 77 Compound 94

The biaryl intermediate was prepared by reaction of 23 with2-amino-4-cyclopropyl-1,3,4-thiadiazole (prepared as in the synthesis of24) using Suzuki coupling B. Deprotection was accomplished by brieftreatment of the protected biaryl intermediate with 90/10 TFA/H₂O anddichloroacetylation was performed as in the synthesis of 29. ¹H NMR (300MHz, CD₃OD): δ 1.12-1.31 (m, 4H), 2.46-2.52 (m, 1H), 4.30-4.75 (m, 3H),5.02 (d, 1H, J=3.3), 6.24 (s, 1H), 7.54 (d, 2H, J=8.4), 7.87 (d, 2H,J=8.4).

Example 78 Compound 95

Same procedure as in the synthesis of 94 using methyl difluoroacetate inplace of methyl dichloroacetate. ¹H NMR (300 MHz, CD₃OD): δ 1.13-1.15(m, 2H), 1.28-1.32 (m, 2H), 2.47-2.52 (m, 1H), 4.29-4.70 (m, 3H), 5.01(d, 1H, J=4.2); 5.98 (t, 1H, J=53.7), 7.54 (d, 2H, J=8.7), 7.88 (d, 2H,J=8.7). LRMS (ESI⁻) m/z: 370.0 (M−H⁺ C₁₆H₁₅F₃N₃O₂S requires 370.1).

Example 79 Compound 97

Compound 96 was prepared in analogous fashion to 24. Compound 97 wasprepared in analogous fashion to 48. Suzuki coupling method B was used,followed by deprotection and difluoroacetylation. LRMS (ESI⁻) m/z: 376.0(M−H⁺ C₁₄H₁₃F₃N₃O₂S₂ requires 376.0)

Example 80 Compound 98

Compound 97 (11.1 mg or 0.0294 mmol) was dissolved in 5 mL of CH₂Cl₂,with stirring at room temperature. To this was added m-CPBA (36 mg,0.147 mmol). The mixture was stirred for 23 hours. AttemptedChromatotron (1 mm plate) purification, eluting with 80:20 then 70:30then 50:50 hexanes/EtOAc; failed to remove m-CPBA-related materials.Thus, material from the Chromotron was subjected to C-8 reversed-phaseHPLC, which gave 8.8 mg (0.021 mmol) of 98. ¹H NMR (300 MHz, CD₃OD): δ3.52 (s, 3H), 4.34-4.72 (m, 3H), 5.05 (d, 1H, J=3.6), 5.97 (t, 1H,J=53.9), 7.61 (d, 2H, J=7.61), 8.05 (d, 2H, J=8.1).

Example 81 Compound 100

Compound 99 was prepared in analogous fashion to 24 and 100 was preparedin analagous fashion to 48. Suzuki coupling method B was used followedby deprotection and difluoroacetylation. ¹H NMR (300 MHz, CD₃OD): δ 2.80(s, 3H), 4.30-4.70 (m, 3H), 5.01 (d, 1H, J=3.6), 5.98 (t, 1H, J=53.9),7.55 (d, 2H, J=8.4), 7.91 (d, 2H, J=8.3).

Example 82 Compound 101

Compound 101 was prepared using the same procedure as in the synthesisof 100 but with methyldichloroacetate in place of methyidifluoroacetate.¹H NMR (300 MHz, CD₃OD): δ 2.79 (s, 3H), 4.31-4.75 (m, 3H), 5.03 (d, 1H,J=3.0), 6.24 (s, 1H), 7.55 (d, 2H, J=8.4), 7.89 (d, 2H, J=8.4).

Example 83 Compound 102

Compound 17 (100 mg, 0.258 mmol) was placed in a dry round-bottom flaskequipped with a stir bar. The flask was charged with 5 mL of dry THF andthe contents cooled to −78° C. under N₂. With rapid stirring, n-BuLi(1.30 M in hexanes, 0.322 mmol, 0.248 mL) was added, which produced aclear brown/yellow solution. The mixture was stirred for an additional10 minutes. Excess CO₂, generated by sublimation of dry ice, was passedthough a drying tube charged with Drierite and bubbled directly into the−78° C. mixture, now equipped with a venting needle in the septum toprevent buildup of CO₂ pressure. The mixture was warmed to roomtemperature and stirred an additional 30 minutes by which time it wasclear yellow and TLC indicated a product had formed. The mixture wasquenched by addition of 10% (v/w) aqueous NH₄Cl (5 drops), producing acloudy yellow suspension. The mixture was concentrated under vacuum andre-suspended in EtOAc (50 mL) and enough 10% aqueous citric acid toproduce a biphasic mixture having a pH of 2.5. The layers were separatedand the EtOAc layer was washed with brine (1×10 mL). The mixture wasdried over Na₂SO₄, filtered and evaporated to give 105 mg of yellow oil.The product was isolated by chromatotron (1 mm plate eluting with 4:1hexanes/EtOAc to 65:35 hexanes/EtOAc to 1:1 hexanes/EtOAc to 2% MeOH inCH₂Cl₂ to 5% MeOH in CH₂Cl₂ and, finally, 10% MeOH in CH₂Cl₂ to whichseveral drops of acetic acid had been added). The appropriate fractionswere combined, diluted with toluene and evaporated to give 67.5 mg(0.191 mmol) of 102. ¹H NMR (300 MHz, CD₃OD): δ 1.49 (s, 9H), 1.57 (S,3H), 1.69 (s, 3H), 3.77-3.90 (m, 1H), 4.39-4.57 (m, 1H), 4.90-5.10 (brs, 1H), 5.18 (d, 1H, J=7.2), 7.56 (d, 2H, J=8.1), 8.04 (d, 2H, J=8.1).LRMS (ESI⁻) m/z: 352 (M−H⁺ C₁₈H₂₃FNO₅ requires 352).

Example 84 Compound 106

Compound 102 (106 mg, 0.299 mmol) was dissolved in EtOAc anddiazomethane, generated using an Aldrich Chemical Company diazomethanekit, was added dropwise until a slight yellow color persisted. Excess ofdiazomethane was allowed to evaporate overnight in a well-ventilatedfume hood, and the remaining EtOAc solution was concentrated undervacuum. The residue was dissolved in Et₂O and washed with aqueous NaHCO₃and then brine. The layers were separated and the organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated to give 0.087 g(0.24 mmol, 79%) of methyl ester 103, which was used without furtherpurification or characterization.

Compound 103 (0.037 g, 0.136 mmol) was dissolved in 1 mL of EtOH. Tothis was added hydrazine hydrate (0.007 g, 0.177 mmol). The mixture wasrefluxed for 12 hours. The solvent was removed under vacuum and theproduct purified by chromatotron (1 mm plate, 10% MeOH in CH₂Cl₂).Compound 104 was isolated (40:0 mg, 0.109 mmol) and was used withoutfurther characterization. LRMS (ESI⁻) m/z: 366 (M−H⁺ C₁₈H₂₅FN₃O₄requires 366).

Compound 104 (0.050 g, 0.133 mmol) was dissolved in 5 mL of triethylorthoformate and stirred at 120° C. for 24 hours. The mixture wasevaporated to give 26 mg of crude 105, which was used without furthercharacterization or purification.

Compound 105 (0.0051 g, 0.014 mmol) was stirred at room temperature with10 mL of 9:1 TFA/H₂O for 10 minutes. The mixture was concentrated undervacuum, dissolved three times in a MeOH/toluene mixture, the solventsbeing evaporated each time, and dried to a constant weight to give 0.5mg of deprotected material. The residue was dissolved in 1 mL of MeOH inan open container and 5 drops of methyl difluoroacetate and 15 drops ofEt₃N were added. The mixture was stirred at room temperature for 12hours. After 12 hours, TLC indicated a single product. The mixture wasconcentrated under vacuum and the product isolated by silica gelchromatography eluting with 20:1 CH₂Cl₂/CH₃OH to give 2.3 mg of 106. ¹HNMR (300 MHz, CD₃OD): δ 4.31-4.74 (m, 3H), 5.04 (d, 1H, J=3.9), 5.96 (t,1H, J=53.9), 7.61 (d, 2H, J=8.4), 8.05 (d, 2H, J=8.4), 8.99 (d, 1H).LRMS (ESI⁻) m/z: 314 (M−H⁺ C₁₃H₁₁F₃N₃O₃ requires 314).

Example 85 Compound 107

Compound 107 was prepared in analogous manner to 106 using methyldichloroacetate in place methyl difluoroacetate. ¹H NMR (300 MHz,CD₃OD): δ 4.32-4.80 (m, 3H), 5.06 (d, 1H, J=3.0), 6.23 (S, 1H), 7.62 (d,2H, J8.4), 8.03 (d, 2H, J=8,4), 8.98 (s. 1H). LRMS (ESI⁻) m/z: 346 (M−H⁺C₁₃H₁₁Cl₂FN₃O₃ requires 346).

Example 86 Compound 108

Compound 102 (32.7 mg, 0.0925 mmol) was dissolved in EtOAc (8 mL) andcooled with stirring to 0° C. Pentafluorophenol (17 mg, 0.093 mmol) wasadded. Once all solids had dissolved, DCC (19 mg, 0.093 mmol) was addedand the mixture stirred at 0° C. for 1.25 hours. The mixture wasevaporated to one-quarter the original volume at which time a DCUprecipitate formed. The DCU was removed by filtration and thepentafluorophenyl ester was isolated by evaporation. The residue wasdissolved in 4 mL of MeOH and cooled to 0° C. with stirring. NaBH₄ (18mg, 0.46 mmol) was added portionwise. When bubbling ceased, the mixturewas warmed to room temperature. After 2 hour, excess NaBH₄ was quenchedby addition of 4 drops of glacial HOAc. The mixture was evaporated todryness and the residue partitioned between 1N aq. HCl and EtOAc. TheEtOAc was separated and washed with 1 N aqueous HCl (2×15 mL), saturatedaqueous NaHCO₃ (2×10 mL) and finally brine (1×25 mL). The EtOAc layerwas dried over anhydrous Na₂SO₄, filtered, and evaporated to give 50 mgof yellow oil. Chromatotron purification (1 mm plate, eluting with 5%EtOAc in hexanes to 10% EtOAc in hexanes) gave 108 (10.3 mg, 0.030mmol). ¹H NMR (300 MHz, CD₃OD): δ 1.49 (s, 9H), 1.57 (s, 3H), 1.68 (s.3H), 3.75-3.89 (m, 1H), 3.90 (s, 2H), 4.39-4.58 (m, 1H), 4.90-5.05 (brs, 1H), 5.19 (d, 1H, J=7.2), 7.57 (d, 2H, J=8.3), 8.04 (d, 2H, J=8.3).

Example 87 Compound 109

Compound 108 (10 mg, 0.30 mmol) was dissolved in 5 mL of 9:1 TFA/H₂O andstirred at room temperature for 1 hour. The mixture was thenconcentrated under vacuum to give approximately 9 mg of deprotectedmaterial as the TFA salt. ¹H NMR (300 MHz, CD₃OD): δ 3.54-3.68 (m, 1H),3.91 (s, 2H), 4.24-4.67 (m, 2H), 4.90 (d, 1H, solvent obscured), 7.57(d, 2H, J=8.4), 8.07 (d, 2H, J=8.4).

The 9 mg of material (0.03 mmol) were dissolved in 2 mL of MeOH in anopen container. To this was added 15 drops of Et₃N and 15 drops ofmethyl dichloroacetate. The mixture was stirred for 56 hour at roomtemperature. By the end of the reaction the solvent had completelyevaporated. The residue was loaded onto a chromatotron (1 mm plate,eluting with 2% MeOH in CH₂Cl₂ to 4% MeOH in CH₂Cl₂, providing 7.5 mg of109. ¹H NMR (300 MHz, CD₃OD): δ 3.88 (s, 2H), 4.28-4.73 (m, 3H), 5.02(d, 1H, J=3.3), 6.22 (s, 1H), 7.51 (d, 2H, J=8.4), 7.97 (d, 2H, J=8.4).

Example 88 Compound 110

Prepared in analogous fashion to other pyridine-containing biarylcompounds herein. ¹H NMR (300 MHz, CD₃OD): δ 2.54 (s, 3H), 4.30-4.68 (m,3H), 4.99 (d, J=3.3), 5.98 (t, 1H, J=40.5), 7.35 (d, 1H, J=6.0), 7.49(d, 2H, J=6.2), 7.61 (d, 2H, J=6.2), 7.95 (dd 1H, J=6.0, 1.7), 8.61 (d,1H, J=1.7). LRMS (ESI⁻) m/z: 336.9 (M−H⁺ C₁₇H₁₆F₃N₂O₂ requires 337.1).

Example 89 Compound 111

Prepared in analogous fashion to other pyridine-containing biarylcompounds herein. ¹H NMR (300 MHz, CD₃OD): δ 2.56 (s, 3H), 4.30-4.74 (m,3H), 5.01 (d, 1H, J=3.9), 6.27 (s, 1H), 7.36 (d, 1H, J=8.1), 7.56 (d,2H, J=8.4), 7.61 (d, 2H, J=8.4), 7.96 (1H, dd, J=8.1, 2.3), 8.62 (d, 1H,J=2.31).

Example 90 Compound 112

Compound 111 (0.0185 g, 0.0499 mmol) was dissolved in 10 mL CH₂Cl₂ at 0°C. To this was added m-CPBA (0.246 g, 0.0997 mmol). The mixture wasstirred until the ice bath melted and the mixture came to roomtemperature, approximately 12 hours. The mixture was concentrated undervacuum and 112 was isolated by preparative plate silica gelchromatography (15% MeOH in CH₂Cl₂) followed by a silica gel plug (3%MeOH in CH₂Cl₂). LRMS (ESI⁻) m/z: 385 (M−H⁺ C₁₇H₁₆Cl₂FN₂O₃ requires385.1).

Example 91 Compound 113

Compound 113 was synthesized from compound 110 in the same manner 112was synthesized from 111. LRMS (ESI⁻) m/z: 353 (M−H⁺ C₁₇H₁₆Cl₂FN₂O₃requires 353.1).

Example 92 Compound 114

This compound was prepared in the same manner as other biaryls herein.LRMS (ESI⁻) m/z: 324 (M−H⁺ C₁₅H₁₃F₃N₃O₂ requires 324.1).

Example 93 Compound 115

Prepared in same manner as compound 48. The required aryl bromide wasprepared in two steps from 2-amino-1,3,4-thiadiazole by bromination withBr₂ followed by acetylation. LRMS (ESI⁻) m/z: 419 (M−H⁺ C₁₅H₁₄Cl₂FN₄O₃Srequires 419.0).

Example 94 Compound 116

Compound 116 was synthesized as set forth in Schemes 2 and 3. Thephenylserine analog was obtained by condensation of4-bromo-2-fluorobenzaldehyde with glycine. Suzuki coupling method B wasused to couple the bromide intermediate with m-pyridineboronic acid.Deprotection and difluoroacetylation were carried out as describedpreviously herein. LRMS (ESI⁺) m/z: 343 (M+H⁺ C₁₆H₁₅F₄N₂O₂ requires343.1).

Example 95 Compound 117

Same procedure as that used for the synthesis of 116. LRMS (ESI⁺) m/z:375 (M+H⁺ C₁₆H₁₅F₄N₂O₂ requires 375.1).

Example 96 Compound 118

Compound 19 (100 mg, 0.258 mmol), potassium acetate (38 mg, 0.386 mmol),and 2 mL of N,N′-dimethylacetamide (DMAC) were combined in a glasspressure tube. Thiazole (112 mg, 1.31 mmol) was added by syringe, andthe syringe rinsed with an additional 1 mL of DMAC, which was added tothe pressure tube. The mixture was stirred at room temperature whilepurging with N₂ for 10 minutes. Pd(PPh₃)₄ (15 mg, 0.013 mmol) was added,the mixture purged with N₂ for 5 minutes, the tube sealed and heated to150° C. behind a blast shield and held at that temperature for 12 hours.The mixture became dark brown. After cooling to room temperature, thecontents of the tube were filtered through a pad of Celite and thefiltrate evaporated to dryness. The residue was dissolved in 40 mL of3:1 EtOAc/hexanes and washed with H₂O (2×15 mL) and brine (2×15 mL) andthen dried over anhydrous Na₂SO₄. The dried organic layer was filteredto remove the drying agent and concentrated under vacuum to give 109 mgof brown oil. Compound 119 was purified by silica gel chromatographyeluting with 80:20 to 50:50 hexanes/EtOAc (54 mg, 0.14 mmol). LRMS(ESI⁺) m/z: 393.1 (M+H⁺ C₂₀H₂₆FN₂O₃S requires 393.2).

All of compound 119 was dissolved in 9:1 (v/v) TFA/H₂O (7.5 mL) andstirred at room temperature for 30 minutes. The mixture was thenconcentrated under vacuum and the residue dissolved twice in a mixtureof toluene and methanol, the solvents being evaporated each time. Aportion of the product (9.7 mg, 0.027 mmol) was dissolved in 2 mL ofMeOH and 10 drops of Et₃N and 12 drops of methyl difluoroacetate wereadded. The mixture was left open to the air and stirred rapidly for 16hours. The mixture was evaporated to dryness and the residue purified bychromatotron (1 mm plate) eluting with 3% MeOH in CH₂Cl₂ to give 118(6.0 mg, 0.018 mmol. ¹H NMR (300 MHz, CD₃OD): δ 4.27-4.69 (m, 3H),4.97(d, 1H, J=4.2), 5.99 (t, 1H, J=53.9), 7.47 (d, 2H, J=8.3), 7.65 (d,2H, J=8.3), 8.16 (s, 1H), 8.94 (s, 1H), LRMS (ESI⁺) m/z: 331.1 (M+H⁺C₁₄H₁₄F₃N₂O₂S requires 331.1).

Example 97 Compound 120

Compound 120 was prepared in a manner analogous to that used to prepare118 using dichloroacetylation instead of difluoroacetylation. LRMS(ESI⁺) m/z: 363.0 (M+H⁺ C₁₄H₁₄Cl₂FN₂O₂S requires 363.0).

Example 98 Compound 121

The protected biaryl intermediate was synthesized by Suzuki couplingmethod B from 23 and 3-chloro-6-methylpyridazine. Deprotection anddichloroacetylation Were performed as for other compounds of thisinvention. LRMS (ESI⁺) m/z: 372.0 (M+H⁺ C₁₆H₁₇Cl₂FN₃O₂ requires 372.1).

Example 99 Compound 122

Prepared in analogous fashion to 121. LRMS (ESI⁺) m/z: 340.1 (M+H⁺C₁₆H₁₇F₃N₃O₂ requires 340.1).

Example 100 Compound 123

The protected biaryl intermediate was prepared by Suzuki coupling methodB from boronic acid 23 and the appropriate aryl bromide. Thisintermediate was then deprotected by brief treatment with 9:1 (v/v)TFA/H₂O and dichloroacetylated as described for 29. LRMS (ESI⁺) m/z:380.0 (M−H⁺ C₁₇H₁₃Cl₂FN₃O₂ requires 380.0).

Example 101 Compound 124

Prepared in analogous fashion to 123. Difluoroacetylation was performedas with other compounds of this invention. LRMS (ESI⁺) m/z: 348.0 (M+H⁺C₁₇H₁₃F₃N₃O₂ requires 348.1).

Example 102 Compound 125

The biaryl intermediate was prepared from boronic acid 23 using Suzukicoupling. LRMS (ESI⁺) m/z: 392.0 (M−H⁺ C₁₅H₁₃Cl₃FN₃O₂ requires 392.0).

Example 103 Compound 126

The starting nitrile was prepared from 35 by analogy to the preparationof bromo intermediate 19. Treatment with Ph₂P(S)SH gave thethiobenzamide which was converted to the thiobenzamidine intermediate byreaction with dimethylformamide dimethylacetal. Cyclization withhydroxylamine-O-sulfonic acid in methanol/pyridine gave the biarylintermediate. LRMS (ESI⁺) m/z: 364.0 (M−H⁺ C₁₃H₁₂Cl₂FN₃O₂S requires364.0)

Example 104 Compound 127

The phenyllithium intermediate was prepared by treatment of intermediate19 with n-butyllithium in THF at −78° C. The intermediate was reactedwith pyridazine and the mixture of the resulting 2- and 3-positionadducts was oxidized with DDQ. The desired 3-pyridazyl regioisomer wasisolated by chromatography. LRMS (ESI⁺) m/z: 326.0 (M−H⁺ C₁₅H₁₄F₃N₃O₂requires 326.0)

Example 105 Compound 128

Compound 128 was synthesized using the procedure in Example 104. LRMS(ESI⁺) m/z: 358.0 (M−H⁺ C₁₅H₁₄Cl₂FN₃O₂ requires 358.0)

Example 106 Compound 129

The procedure discussed in Example 104 was used. LRMS (ESI⁺) m/z: 358.0(M−H⁺ C₁₅H₁₄Cl₂FN₃O₂ requires 358.0)

Example 107 Compound 130

The procedure of Example 104 was used. LRMS (ESI⁺) m/z: 326.0 (M−H⁺C₁₅H₁₄F₃N₃O₂ requires 326.0)

Example 108 Compound 131

The phenyllithium intermediate was reacted withN-methoxy-N-methylacetamide and the resulting acetophenone was reactedwith dimethylformamide dimethylacetal and hydroxylamine-O-sulfonic acidin methanol in presence of pyridine to give the isoxazole. LRMS (ESI⁺)m/z: 347.0 (M−H⁺ C₁₄H₁₃Cl₂N₂O₃ requires 347.0)

Example 109 Compound 132

Compound 132 was synthesized using the procedure in Example 108. LRMS(ESI⁺) m/z: 315.0 (M−H⁺ C₁₄H₁₃F₃N₂O₃ requires 315.0.

Example 110 Compound 133

The phenyllithium intermediate was reacted with dimethylformamide togive the formyl intermediate which was then reacted with the ylidegenerated from methoxymethyltriphenyl phosphonium bromide. The resultingenol ether was brominated with bromine to give the bromoaldehyde.Cyclization with thiourea gave the aminothiazole. LRMS (ESI⁺) m/z: 378.0(M−H⁺ C₁₄H₁₄Cl₂FN₃O₂S requires 378.0)

Example 111 Compound 134

Compound 134 was synthesized using the procedure in Example 110. LRMS(ESI⁺) m/z: 346.0 (M−H⁺ C₁₄H₁₄F₃N₃O₂S requires 346.0)

Example 112 Compound 135 (Mixture of Diastereoisomers)

Compound 135 was synthesized using the procedure in Example 108. LRMS(ESI⁺) m/z: 331.0 (M−H⁺ C₁₄H₁₃F₂N₂O₃ requires 331.0)

Example 113 Compound 136

The starting material in Example 108 was reacted with Lawesson's reagentand the product was cyclized to the biaryl intermediate withhydroxylamine-O-sulfonic acid in methanol in presence of pyridine. LRMS(ESI⁺) m/z: 363.0 (M−H⁺ C₁₄H₁₃Cl₂FN₂O₂S requires 363.0)

Example 114 Compound 137

Compound 137 was synthesized using the procedure in Example 113. LRMS(ESI⁺) m/z: 331.0 (M−H⁺ C₁₄H₁₃F₃N₂O₂S requires 331.0)

Example 115 Compound 138 (Mixture of Diastereoisomers)

Compound 138 was synthesized using the procedure in Example 113. LRMS(ESI⁺) m/z: 347.0 (M−H⁺ C₁₄H₁₃CIF₂N₂O₂S requires 347.0)

Example 116 Compound 139

Compound 139 was synthesized using the procedure in Example 96 startingfrom the desired enantiomer of intermediate 19. LRMS (ESI⁺) m/z: 331.0(M−H⁺ C₁₄H₁₃F₃N₂O₂S requires 331.0)

Example 117 Compound 140 (Mixture of Diastereoisomers)

Compound 140 was synthesized using the procedure in Example 41. LRMS(ESI⁺) m/z: 348.0 (M−H⁺ C₁₃H₁₂CIF₂N₃O₂S requires 348.0)

Example 118 Compound 141

Intermediate 19 was reacted with trimethylsilylacetylene in presence ofcopper iodide and PdCl₂(PPh₃)₂. The product was desilylated by treatmentwith potassium carbonate in methanol. The cyanoacetylene intermediatewas reacting acetylene with n-butyllithium, which was then reacted withtosylcyanide. Cyclization to the 3-aminoisoxazole was performed bytreatment of the cyanoacetylene with hydroxylamine in ethanol. LRMS(ESI⁺) m/z: 330.0 (M−H⁺ C₁₄H₁₄F₃N₃O₃ requires 330.0)

Example 119 Compound 142

Compound 142 was synthesized using the procedure in Example 118. LRMS(ESI⁺) m/z: 362.0 (M−H⁺ C₁₄H₁₄Cl₂FN₃O₃ requires 362.0)

Example 120 Compound 143

The desired enantiomer of intermediate 19 was reacted withbis(pinacolato)diboron in presence of1,1′-bis(diphenylphosphino)ferrocenepalladium (II) dichloridedichloromethane. The pinacolboronate ester was coupled with5-bromo-2-cyanopyridine using the Suzuki reaction. LRMS (ESI⁺) m/z:350.0 (M−H⁺ C₁₇H₁₄F₃N₃O₂ requires 350.0)

Example 121 Compound 144

Compound 144 was synthesized using the procedure in Example 120. LRMS(ESI⁺) m/z: 382.0 (M−H⁺ C₁₇H₁₄Cl₂FN₃O₂ requires 382.0)

Example 122 Compound 145 (Mixture of Diastereoisomers)

Intermediate 19 was reacted with n-butyllithium and then withdimethylformamide. The formyl intermediate was reacted withmethylmagnesium bromide to give the benzylic alcohol as a mixture ofdiastereisomers. LRMS (ESI⁺) m/z: 324.0 (M−H⁺ C₁₃H₁₆Cl₂FNO₃ requires324.0)

Example 123 Compound 146

The starting acetylene, prepared as described in Example 118, wasreacted with n-butyllithium followed by addition of carbon dioxide andthe resulting acid was esterified with diazomethane in ethylacetate/diethyl ether. The 3-hydroxyisoxazole was obtained by reactingthe ester with hydroxylamine and was then methylated with diazomethanein ethyl acetate/diethyl ether. LRMS (ESI⁺) m/z: 377.0 (M−H⁺C₁₅H₁₅Cl₂FN₂O₄ requires 377.0)

Example 124 Compound 147

Compound 147 was synthesized by the method of Example 120 using2-bromo-1,3,4-thiadiazole in a Suzuki coupling. LRMS (ESI⁺) m/z: 364.0(M−H⁺ C₁₃H₁₂Cl₂FN₃O₂S requires 364.0)

Example 125 Compound 148

The starting acetylene was obtained as described in Example 118. The3-carboethoxy isoxazole intermediate was obtained by cycloaddition ofthe nitrile oxide generated in situ from ethyl nitroacetate,di-t-butyldicarbonate and 4-dimethylaminopyridine. The amide wasobtained from the 3-carboethoxy isoxazole intermediate by hydrolysis tothe acid, conversion to the acyl chloride and reaction with ammonia.LRMS (ESI⁺) m/z: 358.0 (M−H⁺ C₁₅H₁₄F₃N₃O₄ requires 358.0)

Example 126 Compound 149

Compound 149 was synthesized using the procedure in Example 108 startingfrom the desired enantiomer of intermediate 19. LRMS (ESI⁺) m/z: 347.0(M−H⁺ C₁₄H₁₃Cl₂FN₂O₃ requires 347.0)

Example 127 Compound 150

The 3-carboxyisoxazole intermediate obtained as described in Example 125was converted into the acid chloride using Vilsmeyer reagent indimethylformamide. The crude product was reduced to the 3-hydroxymethylisoxazole intermediate with tetrabutylammonium borohydride in THF. LRMS(ESI⁺) m/z: 377.0 (M−H⁺ C₁₅H₁₅Cl₂FN₂O₄ requires 377.0)

Example 128 Compound 151

Compound 151 was synthesized using the procedure in Example 127. LRMS(ESI⁺) m/z: 345.0 (M−H⁺ C₁₅H₁₅F₃N₂O₄ requires 345.0)

Example 129 Compound 152

Compound 152 was synthesized using the procedure in Example 125. LRMS(ESI⁺) m/z: 390.0 (M−H⁺ C₁₅H₁₄Cl₂FN₃O₄ requires 390.0)

Example 130 Compound 153

Compound 153 was synthesized using the procedure in Example 40. EDC/HOBTwas used to acylate the amino intermediate with cyanoacetic acid. LRMS(ESI⁺) m/z: 314.0 (M−H⁺ C₁₇H₁₆FN₃O₂ requires 314.0)

Example 131 Compound 154

Compound 154 was synthesized using the procedure in Example 130. LRMS(ESI⁺) m/z: 330.0 (M−H⁺ C₁₆H₁₆FN₅O₂ requires 330.0)

Example 132 Compound 155

The starting acetylene was obtained as described in Example 118. The3-methyl isoxazole intermediate was obtained by cycloaddition of thenitrile oxide generated in situ from nitroethane, di-t-butyldicarbonateand 4-dimethylaminopyridine. LRMS (ESI⁺) m/z: 329.0 (M−H⁺ C₁₅H₁₅F₃N₂O₃requires 329.0)

Example 133 Compound 156

Compound 156 was synthesized using the procedure in Example 132. LRMS(ESI⁺) m/z: 361.0 (M−H⁺ C₁₅H₁₅Cl₂FN₂O₃ requires 361.0)

Example 134 Compound 157

Intermediate 19 was reacted with 4-methylimidazole in refluxingdimethylformamide in the presence of copper powder. LRMS (ESI⁺) m/z:3460.0 (M−H⁺ C₁₄H₁₄Cl₂FN₃O₂ requires 346.0)

Example 135 Compound 158

Compound 158 was synthesized using the procedure in Example 134. LRMS(ESI⁺) m/z: 328.0 (M−H⁺ C₁₅H₁₆F₃N₃O₂ requires 328.0)

Example 136 Compound 159

Compound 159 was synthesized using the procedure in Example 126. LRMS(ESI⁺) m/z: 315.0 (M−H⁺ C₁₄H₁₃F₃N₂O₃ requires 315.0)

Example 137 Compound 160

A mixture of the desired enantiomer of intermediate 19 (1 g, 2.5 mmol),potassium carbonate (712 mg, 5 mmol), and propiolamide (1.07 g, 15 mmol)in N,N-dimethylacetamide (4 mL) was stirred at room temperature whilepurging with N₂ for 10 minutes. Pd(PPh₃)₄ catalyst (15 mg, 0.013 mmol)was added and the mixture was purged with N₂ for another 5 minutes. Theresulting mixture was stirred at 100° C. for 9 hrs. After cooling toroom temperature, the crude was flash silica gel chromatographed to givethe coupling product (601 mg). ¹H NMR (300 MHz, CDCl₃): δ 1.49 (s, 9H),1.59 (s, 3H), 1.70 (s, 3H), 3.75-3.90 (m, 1H), 4.35-4.60 (m, 2H), 5.15(d, 1H, J=7.5), 5.65 (s, 1H), 5.85 (s, 1H), 7.45 (d, 2H, J=8.4), and7.55 (d, 2H, J=8.4).

To dimethylformamamide (2 mL) was slowly added thionyl chloride (2 mL)at 0° C. and the reaction mixture was stirred at that temperature for 30min. The solution was then cannulated to a solution of the propiolamideintermediate (600 mg) in DMF (4 mL) at 0° C. After stirring at roomtemperature for 30 min, the mixture was poured into ice/water (50 mL).The pH was adjusted to 7 with saturated sodium bicarbonate. The solutionwas extracted with ethyl acetate three times. The combined organiclayers were washed with water, dried over anhydrous sodium sulfate, andpurified by column chromatography to give propiolonitrile intermediate(326 mg). ¹H NMR (300 MHz, CDCl₃): δ 1.49 (s, 9H), 1.59 (s, 3H), 1.70(s, 3H), 3.75-3.90 (m, 1H), 4.35-4.60 (m, 2H), 5.15 (d, 1H, J=7.5), 7.49(d, 2H, J=8.4), and 7.63 (d, 2H, J=8.4).

To a solution of sodium hydroxide (206 mg, 7 eq.) in water (2 mL), wasadded hydroxylamine hydrochloride (256 mg, 5 eq.). The solution wasadded to the propiolonitrile intermediate (263 mg) dissolved in ethanol(7 mL). The resulting mixture was stirred at room temperature for 6 hrs.Ethyl acetate was added, the organic layer was separated, washed withwater, and dried with anhydrous sodium sulfate. The crude material waspurified by column chromatography to give the protected 3-aminoisoxazolewhich was then dissolved in THF (6 mL) and 4 N HCl (6 mL), and stirredat 80° C. for 2.5 hrs. The reaction mixture was evaporated to drynessunder reduced pressure. The residue was co-evaporated with methanoltwice and dried overnight under vacuum. To the crude material dissolvedin methanol (2 mL) was added triethylamine (2 mL), followed by methyldichloroacetate (1 mL). The mixture was stirred at room temperature for24 hrs. The solvent was evaporated and the mixture was purified by flashcolumn chromatography to give compound 160 (167 mg). ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 4.35-4.70 (m, 3H), 5.05 (m, 1H), 5.85 (s, 1H), 6.16 (s,1H), 7.44 (d, 2H, J=8.1), and 7.65 (d, 2H, J=8.1). LRMS (ESI⁺) m/z:362.0 (M−H⁺ C₁₄H₁₄Cl₂FN₃O₃ requires 362.0)

Example 138 Compound 161

Compound 161 was synthesized using the procedure in Example 132 startingfrom the desired enantiomer of intermediate 19. LRMS (ESI⁺) m/z: 361.0(M−H⁺ C₁₅H₁₅Cl₂FN₂O₃ requires 361.0)

Example 139 Compound 162

Compound 162 was synthesized using the procedure in Example 138. LRMS(ESI⁺) m/z: 329.0 (M−H⁺ C₁₅H₁₅F₃N₂O₃ requires 329.0)

Example 140 Compound 163

Compound 163 was synthesized using the procedure in Example 120.Dipyclohexylcarbodiimide was used to acylate the amino intermediate withcyanoacetic acid. LRMS (ESI⁺) m/z: 339.0 (M−H⁺ C₁₈H₁₅FN₄O₂ requires339.0)

Example 141 Compound 164

Compound 164 was synthesized using the procedure in Example 120.Dicyclohexylcarbodiimide was used to acylate the amino intermediate withazidoacetic acid. LRMS (ESI⁺) m/z: 355.0 (M−H⁺ C₁₇H₁₅FN₆O₂ requires355.0)

Example 142 Compound 165

Compound 165 was synthesized using the procedure in Example 120. Theamino intermediate was acetylated with acetic anhydride. LRMS (ESI⁺)m/z: 314.0 (M−H⁺ C₁₇H₁₆FN₃O₂ requires 314.0)

Example 143 Compound 166

Compound 166 was synthesized using the procedure in Example 120. LRMS(ESI⁺) m/z: 350.0 (M−H⁺ C₁₃H₁₁F₄N₃O₂S requires 350.0)

Example 144 Compound 167

Compound 167 was synthesized using the procedure in Example 120.Dicyclohexylcarbodiimide was used to acylate the amino intermediate withN-Boc-glycine and the resulting glycinamide was deprotected withhydrochloric acid in methanol. LRMS (ESI⁺) m/z: 329.0 (M−H⁺ C₁₇H₁₇FN₄O₂requires 329.0)

Example 145 Compound 168

Compound 168 was synthesized using the procedure in Example 144. LRMS(ESI⁺) m/z: 343.0 (M−H⁺ C₁₈H₁₉FN₄O₂ requires 343.0)

Example 146 Compound 169

The desired enantiomer of intermediate 19 (1.16 g, 3 mmol), potassiumcarbonate (1.659 g, 12 mmol), imidazole (1.225 g, 18 mmol), and copperpowder (191 mg, 3 mmol) in 20 mL of DMF were stirred vigorously atreflux for 4 hrs. The reaction mixture was cooled to room temperatureand poured into water (100 mL). After extraction with ethyl acetate andwashing of the organic extract with water (3×), the organic layer waspassed through a silica gel and anhydrous sodium sulfate plug.Evaporation of solvent gave the imidazole (1.06 g). ¹H NMR (300 MHz,CDCl₃): δ 1.45 (s, 9H), 1.60 (s, 3H), 1.75 (s, 3H), 3.75-3.85 (m, 1H),4.35-4.55 (m, 2H), 5.20 (d, 1H, J=7.5), 7.22 (s, 1H), 7.28 (s, 1H), 7.41(d, 2H, J=8.4), 7.58 (d, 2H, J=8.4), and 7.89 (s, 1H).

The imidazole intermediate (2.77 g) was dissolved in THF (20 mL) and 4 NHCl (20 mL), and stirred at 80° C. for 2.5 hrs. The reaction mixture wasevaporated to dryness under reduced pressure. The residue wasco-evaporated with methanol twice and dried overnight under vacuum. Thecrude material was dissolved in methanol (20 mL) and triethylamine (5mL) was added, followed by methyl dichloroacetate (5 mL). The mixturewas stirred at room temperature for 20 hrs. The solvent was evaporatedand the mixture was purified by flash column chromatography to givecompound 169 (1.1 g) as white solid. ¹H NMR (300 MHz, CDCl₃): δ4.30-4.75 (m, 3H), 5.21 (d, 1H, J=7.5), 5.86 (s, 1H), 7.05 (d, 1H,J=7.5), 7.10 (s, 1H), 7.28 (s, 1H), 7.39 (d, 2H, J=8.4), 7.52 (d, 2H,J=8.4 Hz), and 7.81 (s, 1H). LRMS (ESI⁺) m/z: 360.0 (M−H⁺ C₁₅H₁₆C₁₂FN₃O₂requires 360.0)

Example 147 Compound 170

Compound 170 was synthesized by the method of Example 106 using thedesired enantiomer of intermediate 19. LRMS (ES⁺) m/z: 358.0 (M−H⁺C₁₅H₁₄Cl₂FN₃O₂ requires 358.0)

Example 148 Compound 171

Compound 171 was synthesized using the procedure in Example 147. LRMS(ESI⁺) m/z: 326.0 (M−H⁺ C₁₅H₁₄F₃N₃O₂ requires 326.0)

Example 149 Compound 172

Compound 172 was synthesized by the method of Example 110 using thedesired enantiomer of intermediate 19. LRMS (ESI⁺) m/z: 346.0 (M−H⁺C₁₄H₁₄F₃N₃O₂S requires 346.0)

Example 150 Compound 173

Compound 173 was synthesized using the procedure in. Example 137. LRMS(ESI⁺) m/z: 330.0 (M−H⁺ C₁₄H₁₄F₃N₃O₃ requires 330.0)

Example 151 Compound 174

Compound 174 was synthesized using the procedure in Example 146. LRMS(ESI⁺) m/z: 314.0 (M−H⁺ C₁₄H₁₄F₃N₃O₂ requires 314.0)

Example 152 Compound 175

Compound 175 was synthesized using the procedure in Example 28. LRMS(ESI⁺) m/z: 290.0 (M−H⁺ C₁₃H₁₄F₃NO₃ requires 290.0)

Example 153 Compound 176

Compound 176 was synthesized using the procedure in Example 149. LRMS(ESI⁺) m/z: 378.0 (M−H⁺ C₁₄H₁₄C₁₂FN₃O₂S requires 378.0)

Example 154 Compound 177

Compound 177 was synthesized by the method of Example 33 using thebromopyridine derivative and boronic acid 23. LRMS (ESI⁺) m/z: 372.0(M−H⁺ C₁₆H₁₆C₁₂FN₃O₂ requires 372.0)

Example 155 Compound 178

Compound 178 was synthesized by the method of Example 33 using abromopyrimidine derivative and boronic acid 23. LRMS (ESI⁺) m/z: 373.0(M−H⁺ C₁₅H₁₅Cl₂FN₄O₂ requires 373.0)

Example 156 Compound 179

Compound 179 was synthesized by the method of Example 33 using thebromopyrimidine derivative and boronic acid 23. Standard conditions wereused for the removal of protecting groups and the introduction ofdichloroacetamide functionality. LRMS (ESI⁺) m/z: 373.0 (M−H⁺C₁₅H₁₅Cl₂FN₄O₂ requires 373.0)

Example 157 Compound 180

Compound 180 was synthesized by the method of Example 33 using therequisite chloropyridazine derivative and boronic acid 23. Standardconditions were used for the removal of protecting groups and theintroduction of dichloroacetamide functionality. LRMS (ESI⁺) m/z: 373.0(M−H⁺ C₁₅H₁₅Cl₂FN₄O₂ requires 373.0)

Conclusion

Thus, it will be appreciated that the present invention provides novelflorfenicol-like compounds and methods for their use in the treatment orprevention of bacterial infection in animals or humans.

Although certain embodiments and examples have been used to describe thepresent invention, it will be apparent to those skilled in the art thatchanges in the embodiments and examples shown may be made withoutdeparting from the scope of this invention.

1-25. (canceled)
 26. A method of preventing a bacterial infection in anonhuman patient comprising administering to the nonhuman patient apharmaceutically effective amount of a compound having the chemicalformula:

wherein R² and R³ are independently selected from the group consistingof hydrogen, (1C-4C)alkyl, halo, —CF₃, —NH₂, —CN and N₃; wherein R⁴ isselected from the group consisting of:

wherein A¹ is carbon or nitrogen, and carbon atoms in the ring areindependently substituted with an entity selected from the groupconsisting of hydrogen, (1C-4C)alkyl, (3C-6C)cycloalkyl, (1C-4C)alkylO—,—CF₃, —OH, —CN, halo, (1C-4C)alkylSO—, (1C-4C)alkylSO₂—, NH₂SO₂—,(1C-4C)alkylNHSO₂—, ((1C-4C)alkyl)₂NSO₂—, —NH₂, (1C-4C)alkylNH—,((1C-4C)alkyl)₂N—, (1C-4C)alkylSO₂NH—, (1C-4C)alkylC(O)—,(3C-6C)cycloalkylC(O)—, (1C-4C)alkylOC(O)—, (1C-4C)alkylC(O)NH—,—C(O)NH₂, (1C-4C)alkylNHC(O)— and ((1C-4C)alkyl)₂NC(O)—, wherein any ofthe alkyl groups within the substituents may be unsubstituted orsubstituted with a group selected from halo and hydroxy; wherein A², A³,A⁴, and A⁵ are independently selected from the group consisting ofcarbon, nitrogen, oxygen and sulfur, provided that at least one of A¹-A⁵is not carbon, that the total number of nitrogen, oxygen and sulfuratoms in the ring does not exceed 4 and that the ring is aromatic; andwherein if A¹ is carbon and the ring does not contain oxygen or sulfur,one of the nitrogen atoms may optionally be substituted with an entityselected from the group consisting of (1C-4C)alkyl, (1C-4C)alkylSO₂— and—NH₂; and wherein A⁶, A⁷, A⁸, A⁹ and A¹⁰ are independently selected fromthe group consisting of carbon, nitrogen and

provided that only one of A⁶-A¹⁰ at a time can be

and that one, two, or three of the A⁶-A¹⁰ atoms are nitrogen; andwherein the carbon atoms in the ring are independently substituted withan entity selected from the group consisting of hydrogen, (1C-4C)alkyl,(3C-6C)cycloalkyl, (1C-4C)alkylO—, —CF₃, —OH, —CN, halo,(1C-4C)alkylSO—, (1C-4C)alkylSO₂—, NH₂SO₂—, (1C-4C)alkylNHSO₂—,((1C-4C)alkyl)₂NSO₂—, —NH₂, (1C-4C)alkylNH—, ((1C-4C)alkyl)₂N—,(1C-4C)alkylSO₂NH—, (1C-4C)alkylC(O)—, (3C-6C)cycloalkylC(O)—,(1C-4C)alkylOC(O)—, (1C-4C)alkylC(O)NH—, —C(O)NH₂, (1C-4C)alkylNHC(O)—,((1C-4C)alkyl)₂NC(O)— and —OCH₂O—, wherein the oxygen atoms with the—OCH₂O— substituent being bonded to adjacent ring carbon atoms, andwherein any of the alkyl groups within any of the substituents may beunsubstituted or substituted with a group selected from halo andhydroxy; and wherein R⁸ is hydrogen in all compounds, except when R² andR³ are both F, in which case R⁸ is hydrogen or F; and, the compound iseither a racemate having the relative stereochemistry shown or issubstantially enantiomerically pure and has the absolute stereochemistryshown.
 27. The method of claim 26, wherein R² and R³ are independentlyselected from the group consisting of Cl and F; and wherein R⁸ ishydrogen.
 28. The method of claim 27, wherein: R⁴ is

and, wherein any of A⁶-A¹⁰ that is a carbon atom is substituted with anentity selected from the group consisting of hydrogen, —NH₂, halo-, —CN,(1C-4C)alkyl-, (1C-4C)alkylC(O)—, (1C-4C)alkylSO—, (1C-4C)alkylSO₂,NH₂SO₂—, (1C-4C)alkylSO₂NH—, (1C-4C)alkylNHSO₂—, ((1C-4C)alkyl)₂NSO₂—,wherein any of the alkyl groups within any of the substituents may beunsubstituted or substituted with halo or hydroxy.
 29. The method ofclaim 27, wherein R⁴ is selected from the group consisting of:


30. The method of claim 27 wherein R⁴ is


31. The method of claim 30, wherein all carbon atoms and nitrogen atomsare unsubstituted.
 32. The method of claim 30, wherein one of the A²-A⁵atoms that is carbon is substituted with an —NH₂ group, and all othercarbon and nitrogen atoms in the ring are unsubstituted.
 33. The methodof claim 30, wherein R⁴ is selected from the group consisting of:


34. The method of claim 27 selected from the group consisting of:

wherein the compound is either a racemate having the relativestereochemistry shown or is substantially enantiomerically pure and hasthe absolute stereochemistry shown.
 35. The method of claim 34 selectedfrom the group consisting of:

wherein the compound is either a racemate having the relativestereochemistry shown or is substantially enantiomerically pure and hasthe absolute stereochemistry shown.
 36. The method of claim 26, wherein:R⁴ is

and, wherein any of A⁶-A¹⁰ that is a carbon atom is substituted with anentity selected from the group consisting of hydrogen, —NH₂, halo-, —CN,(1C-4C)alkyl-, (1C-4C)alkylC(O)—, (1C-4C)alkylSO—, (1C-4C)alkylSO₂,NH₂SO₂—, (1C-4C)alkylSO₂NH—, (1C-4C)alkylNHSO₂—, ((1C-4C)alkyl)₂NSO₂—,wherein any of the alkyl groups within any of the substituents may beunsubstituted or substituted with halo or hydroxy.
 37. The method ofclaim 26, wherein: R⁴ is

wherein any of A⁶-A¹⁰ that are carbon atoms are optionally substitutedwith —NH₂, and wherein all remaining A⁶-A¹⁰ carbon atoms areunsubstituted.
 38. The method of claim 26, wherein R⁴ is selected fromthe group consisting of:


39. The method of claim 26, wherein R⁴ is


40. The method of claim 39, wherein all carbon atoms and nitrogen atomsare unsubstituted.
 41. The method of claim 39, wherein one of the A²-A⁵atoms that is carbon is substituted with an —NH₂ group, and all othercarbon and nitrogen atoms in the ring are unsubstituted.
 42. The methodof claim 39, wherein R⁴ is selected from the group consisting of:


43. The method of claim 26, selected from the group consisting of:

wherein the compound is either a racemate having the relativestereochemistry shown or is substantially enantiomerically pure and hasthe absolute stereochemistry shown.
 44. A method of preventing abacterial infection in a nonhuman patient comprising administering tothe nonhuman patient a pharmaceutically effective amount of a compoundhaving the chemical formula:


45. The method of claim 44, wherein the compound is a racemate havingthe relative stereochemistry shown.
 46. The method of claim 44, whereinthe compound is substantially enantiomerically pure and has a1-(R)-2-(S) absolute configuration.
 47. The method of claim 26, whereinthe bacterial infection is caused by a bacteria of the genusPasteurella, Haemophilus, Fusobacterium, Bacterioides, Aeromonas,Enterobacter, Escherichia, Klebsiella, Salmonella, Shigella,Actinobacillus, Streptococcus, Mycoplasma, Edwardsiella, Staphylococcus,Enterococcus, Bordetella, Proteus, or Mannheimia.
 48. The method ofclaim 47, wherein the bacterial infection is caused by Mannhemiahaemolytica, Pasteurella multocida, Haemophilus somnus, Fusobacteriumnecrophorum, Bacterioides melaninogenicus, Actinobacilluspleuropneumoniae, Streptococcus suis, Salmonella cholerasuis, Mycoplasmabovis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasmagallisepticum, Edwardsiella ictaluri, Escherichia coli, Enterobactercloacae, Staphylococcus aureus, Staphylococcus intermedius, Enterococcusfaecalis, Enterococcus faecium, Klebsiella pneumoniae, Klebsiellaoxytoca, Enterobacter cloacae, Proteus mirabilis, or Aeromonassalmonicida.